Ror1 antibodies

ABSTRACT

There is described Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1) antibodies that specifically bind a ROR1 polypeptide, and their use. In particular, isolated monoclonal antibodies are described and their use in a number of applications, including in the detection, prevention and treatment of cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 16/628,512,filed Jan. 3, 2020, now U.S. Pat. No. 11,466,083, which is the NationalStage Entry of International Application No. PCT/GB2018/051914, filedJul. 5, 2018, which claims the benefit of GB Application No. 1710835.8,filed Jul. 5, 2017, the contents of each of which are herebyincorporated by reference herein in their entirety.

The invention relates to Receptor Tyrosine Kinase Like Orphan Receptor 1(ROR1) antibodies that specifically bind a ROR1 polypeptide, and theiruse.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML file format and is hereby incorporatedby reference in its entirety. Said XML copy, created on Oct. 3, 2022, isnamed 2013607-0005_SL.xml and is 278,845 bytes in size.

BACKGROUND OF THE INVENTION

Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1) (also known asNeurotrophic Tyrosine Kinase, Receptor-Related 1, NTRKR1) is anonco-foetal antigen expressed during embryogenesis but with limitedexpression on normal adult tissue. It is however expressed on a numberof haematological and solid malignancies: Chronic Lymphocytic Leukaemia(CLL), Acute Lymphoblastic Leukaemia (ALL), Mantle Cell Leukaemia, HairyCell Leukaemia, Pancreatic cancer, Prostate cancer, colon cancer,bladder cancer, ovarian cancer, glioblastoma, testicular cancer, uterinecancer, adrenal cancer, breast cancer, lung cancer, melanoma,neuroblastoma, sarcoma, renal cancer. Furthermore, ROR1 is expressed ona subset of cancer stem cells.

As such, ROR1 is an attractive therapeutic target. Furthermore, a needremains for agents that can be used to treat and/or diagnose theaforementioned cancers.

SUMMARY OF THE INVENTION

A first aspect of the invention relates to an isolated monoclonalantibody comprising a light chain variable domain and a heavy chainvariable domain wherein the light chain variable domain comprises alight chain complementarity determining region (LCDR)1, an LCDR2 and anLCDR3, wherein LCDR1 comprises an amino acid sequence set forth in SEQID NO: 30, SEQ ID NO: 24, SEQ ID NO: 9, SEQ ID NO: 37, SEQ ID NO: 44,SEQ ID NO: 50, SEQ ID NO: 56, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO:2, SEQ ID NO: 16, or SEQ ID NO: 22; LCDR2 comprises an amino acidsequence set forth in SEQ ID NO: 32, SEQ ID NO: 26, SEQ ID NO: 11, SEQID NO: 39, SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 58, SEQ ID NO: 4 orSEQ ID NO: 18; and LCDR3 comprises an amino acid sequence set forth inSEQ ID NO: 34, SEQ ID NO: 28, SEQ ID NO: 13, SEQ ID NO: 41, SEQ ID NO:48, SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 65, SEQ ID NO: 68, SEQ IDNO: 74, SEQ ID NO: 6, or SEQ ID NO: 20; and wherein the heavy chainvariable domain comprises a heavy chain complementarity determiningregion (HCDR)1, an HCDR2 and an HCDR3, wherein HCDR1 comprises an aminoacid sequence set forth in SEQ ID NO: 121, SEQ ID NO: 115, SEQ ID NO:96, SEQ ID NO: 127, SEQ ID NO: 134, SEQ ID NO: 140, SEQ ID NO: 146, SEQID NO: 152, SEQ ID NO: 158, SEQ ID NO: 163, SEQ ID NO: 89, SEQ ID NO:103 or SEQ ID NO: 110; HCDR2 comprises an amino acid sequence set forthin SEQ ID NO: 123, SEQ ID NO: 117, SEQ ID NO: 98, SEQ ID NO: 129, SEQ IDNO: 136, SEQ ID NO: 142, SEQ ID NO: 148, SEQ ID NO: 154, SEQ ID NO: 160,SEQ ID NO: 165, SEQ ID NO: 91 or SEQ ID NO: 105; and HCDR3 comprises anamino acid sequences set forth in SEQ ID NO: 248, SEQ ID NO: 252, SEQ IDNO: 100, SEQ ID NO: 131, SEQ ID NO: 138, SEQ ID NO: 144, SEQ ID NO: 150,SEQ ID NO: 156, SEQ ID NO: 162, SEQ ID NO: 167, SEQ ID NO: 93, SEQ IDNO: 107 or SEQ ID NO: 113, wherein the sequence of each complementaritydetermining region may differ from the given sequence at up to two aminoacid positions, and wherein the monoclonal antibody specifically bindsto a ROR1 polypeptide.

Preferably the isolated monoclonal antibody comprises a light chainvariable domain, wherein the light chain variable domain comprises alight chain complementarity determining region (LCDR)1, an LCDR2 and anLCDR3, wherein

-   -   (a) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 30, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 32, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 34;    -   (b) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 24, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 26, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 28;    -   (c) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 9, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 11, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 13;    -   (d) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 37, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 39, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 41;    -   (e) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 44, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 46, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 48;    -   (f) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 50, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 52, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 54;    -   (g) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 56, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 58, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 60;    -   (h) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 44, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 46, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 65;    -   (i) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 67, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 58, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 68;    -   (j) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 71, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 26, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 74;    -   (k) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 2, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 4, and the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 6;    -   (l) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 16, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 18, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 20; or    -   (m) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 22, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 18, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 20;    -   wherein the sequence of each complementarity determining region        may differ from the given sequence at up to two amino acid        positions, and    -   wherein the monoclonal antibody specifically binds to a ROR1        polypeptide.

Preferably the isolated monoclonal antibody comprises a heavy chainvariable domain, wherein the heavy chain variable domain comprises aheavy chain complementarity determining region (HCDR)1, an HCDR2 and anHCDR3, wherein

-   -   (a) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 121, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 123, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 248;    -   (b) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 115, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 117, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 252;    -   (c) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 96, the HCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 98, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 100;    -   (d) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 127, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 129, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 131;    -   (e) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 134, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 136, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 138;    -   (f) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 140, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 142, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 144;    -   (g) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 146, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 148, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 150;    -   (h) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 152, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 154, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 156;    -   (i) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 158, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 160, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 162;    -   (j) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 163, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 165, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 167;    -   (k) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 89, the HCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 91, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 93;    -   (l) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 103, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 105, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 107; or    -   (m) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 110, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 105, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 113;    -   wherein the sequence of each complementarity determining region        may differ from the given sequence at up to two amino acid        positions, and    -   wherein the monoclonal antibody specifically binds to a ROR1        polypeptide.

More preferably, the isolated monoclonal antibody comprises a lightchain variable domain and a heavy chain variable domain, wherein thelight chain variable domain comprises a light chain complementaritydetermining region (LCDR)1, an LCDR2 and an LCDR3 and the heavy chainvariable domain comprises a heavy chain complementarity determiningregion (HCDR)1, an HCDR2 and an HCDR3, wherein

-   -   (a) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 30, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 32, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 34, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 121, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 123, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 248;    -   (b) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 24, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 26, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 28, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 115, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 117, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 252;    -   (c) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 9, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 11, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 13, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 96, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 98, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 100;    -   (d) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 37, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 39, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 41, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 127, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 129, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 131;    -   (e) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 44, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 46, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 48, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 134, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 136, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 138;    -   (f) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 50, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 52, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 54, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 140, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 142, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 144;    -   (g) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 56, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 58, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 60, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 146, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 148, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 150;    -   (h) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 44, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 46, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 65, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 152, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 154, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 156;    -   (i) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 67, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 58, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 68, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 158, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 160, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 162;    -   (j) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 71, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 26, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 74, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 163, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 165, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 167;    -   (k) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 2, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 4, the LCDR3 comprises the amino acid sequence set        forth as SEQ ID NO: 6, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 89, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 91, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 93;    -   (l) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 16, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 18, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 20, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 103, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO:105, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 107;        or    -   (m) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 22, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 18, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 20, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 110, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 105, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 113;    -   wherein the sequence of each complementarity determining region        may differ from the given sequence at up to two amino acid        positions, and    -   wherein the monoclonal antibody specifically binds to a ROR1        polypeptide.

A second aspect of the invention relates to an isolated monoclonalantibody comprising a light chain variable domain wherein the lightchain variable domain comprises a light chain complementaritydetermining region (LCDR)1, an LCDR2 and an LCDR3, wherein LCDR1comprises an amino acid sequence set forth in SEQ ID NO: 30, SEQ ID NO:24, SEQ ID NO: 9, SEQ ID NO: 37, SEQ ID NO: 44, SEQ ID NO: 50, SEQ IDNO: 56, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 2, SEQ ID NO: 16, orSEQ ID NO: 22; LCDR2 comprises an amino acid sequence set forth in SEQID NO: 32, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 39, SEQ ID NO: 46,SEQ ID NO: 52, SEQ ID NO: 58, SEQ ID NO: 4 or SEQ ID NO: 18; and LCDR3comprises an amino acid sequence set forth in SEQ ID NO: 34, SEQ ID NO:28, SEQ ID NO: 13, SEQ ID NO: 41, SEQ ID NO: 48, SEQ ID NO: 54, SEQ IDNO: 60, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 6, orSEQ ID NO: 20; wherein the sequence of each complementarity determiningregion may differ from the given sequence at up to two amino acidpositions, and wherein the monoclonal antibody specifically binds to aROR1 polypeptide.

A third aspect of the invention relates to an isolated monoclonalantibody comprising a heavy chain variable domain wherein the heavychain variable domain comprises a heavy chain complementaritydetermining region (HCDR)1, an HCDR2 and an HCDR3, wherein HCDR1comprises an amino acid sequence set forth in SEQ ID NO: 121, SEQ ID NO:115, SEQ ID NO: 96, SEQ ID NO: 127, SEQ ID NO: 134, SEQ ID NO: 140, SEQID NO: 146, SEQ ID NO: 152, SEQ ID NO: 158, SEQ ID NO: 163, SEQ ID NO:89, SEQ ID NO: 103 or SEQ ID NO: 110; HCDR2 comprises an amino acidsequence set forth in SEQ ID NO: 123, SEQ ID NO: 117, SEQ ID NO: 98, SEQID NO: 129, SEQ ID NO: 136, SEQ ID NO: 142, SEQ ID NO: 148, SEQ ID NO:154, SEQ ID NO: 160, SEQ ID NO: 165, SEQ ID NO: 91 or SEQ ID NO: 105;and HCDR3 comprises an amino acid sequences set forth in SEQ ID NO: 248,SEQ ID NO: 252, SEQ ID NO: 100, SEQ ID NO: 131, SEQ ID NO: 138, SEQ IDNO: 144, SEQ ID NO: 150, SEQ ID NO: 156, SEQ ID NO: 162, SEQ ID NO: 167,SEQ ID NO: 93, SEQ ID NO: 107 or SEQ ID NO: 113, wherein the sequence ofeach complementarity determining region may differ from the givensequence at up to two amino acid positions, and wherein the monoclonalantibody specifically binds to a ROR1 polypeptide.

The present invention also relates to an isolated antigen bindingfragment of the disclosed antibodies; a composition comprising aneffective amount of the disclosed antibodies or antigen bindingfragments in combination with a pharmaceutically acceptable carrier; andan isolated nucleic acid molecule encoding the disclosed monoclonalantibodies or antigen binding fragments.

The invention further relates to a method of detecting cancer in asubject comprising contacting a biological sample from the subject withat least one of the disclosed isolated monoclonal antibodies or antigenbinding fragments thereof, and detecting antibody bound to the sample,wherein the presence of antibody bound to the sample indicates that thesubject has cancer.

Additionally, the present invention relates to a method for preventingor treating cancer in a subject, comprising administering to the subjecta therapeutically effective amount of at least one of the disclosedantibodies or an antigen binding fragment thereof, a nucleic acidencoding the antibody, and/or a nucleic acid encoding the antigenbinding fragment, thereby preventing or treating cancer, as well as thedisclosed monoclonal antibodies or an antigen binding fragment thereoffor use in the treatment or prevention of cancer.

The disclosed monoclonal antibodies specifically bind to a ROR1polypeptide. In additional embodiments, the monoclonal antibodiesspecifically bind a ROR1 polypeptide with an equilibrium constant(K_(D)) of about 6×10⁻⁹ M or less. In some embodiments, the monoclonalantibodies specifically bind a ROR1 polypeptide with a K_(D) of about1.6×10⁻⁹ M or less, about 2×10⁻⁹ M or less, about 3×10⁻⁹ M or less,about 4×10⁻⁹ M or less or about 5×10⁻⁹ M or less.

The disclosed monoclonal antibodies provide a number of advantages overknown ROR1 antibodies, in particular, those based on clones A and F(more details below). Compared to ROR1 antibodies described in the priorart, the antibodies described herein may have one or more of thefollowing advantages, amongst others: a relatively high binding affinityfor ROR1 (e.g. a low K_(D)), bind to a unique ROR1 epitope, can bind inscFv format, show toxicity on cells expressing ROR1 (e.g. cancer cellssuch as CLL), can be internalised, invoke different cytokine release,show enhanced persistence, and have decreased immunogenicity.

DETAILED DESCRIPTION Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8). Unless otherwise explained,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs.

Administration: The introduction of a composition into a subject by achosen route. Administration can be local or systemic. For example, ifthe chosen route is intravenous, the composition is administered byintroducing the composition into a vein of the subject. In some examplesa disclosed antibody specific for a ROR1 polypeptide, is administered toa subject.

Agent: Any substance or any combination of substances that is useful forachieving an end or result; for example, a substance or combination ofsubstances useful for preventing or treating cancer. Agents include, andare not limited to, proteins, nucleic acid molecules, compounds, smallmolecules, organic compounds, inorganic compounds, or other molecules ofinterest. An agent can include a therapeutic agent (such as ananti-viral agent), a diagnostic agent or a pharmaceutical agent. In someembodiments, the agent is a polypeptide agent (such as a neutralizingantibody). The skilled artisan will understand that particular agentsmay be useful to achieve more than one result.

Amino acid substitution: The replacement of one amino acid in a peptidewith a different amino acid.

Amplification: A technique that increases the number of copies of anucleic acid molecule (such as an RNA or DNA). An example ofamplification is the polymerase chain reaction, in which a biologicalsample is contacted with a pair of oligonucleotide primers, underconditions that allow for the hybridization of the primers to a nucleicacid template in the sample. The primers are extended under suitableconditions, dissociated from the template, and then re-annealed,extended, and dissociated to amplify the number of copies of the nucleicacid. The product of amplification can be characterized byelectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in PCT Publication No. WO 90/01069; ligase chain reactionamplification, as disclosed in European Patent Publication EP-A-320 308;gap filling ligase chain reaction amplification, as disclosed in U.S.Pat. No. 5,427,930; and NASBA™ RNA transcription-free amplification, asdisclosed in U.S. Pat. No. 6,025,134.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: A polypeptide substantially encoded by an immunoglobulin geneor immunoglobulin genes, or antigen binding fragments thereof, whichspecifically binds and recognizes an analyte (antigen) such as a ROR1polypeptide, or an immunogenic fragment thereof. Immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as the myriad immunoglobulin variable regiongenes.

Antibodies exist, for example, as intact immunoglobulins and as a numberof well characterised fragments produced by digestion with variouspeptidases. For instance, Fabs, Fvs, scFvs that specifically bind to aROR1 polypeptide, or fragments of this polypeptide, are specific bindingagents. A scFv protein is a fusion protein in which a light chainvariable region of an immunoglobulin and a heavy chain variable regionof an immunoglobulin are bound by a linker, while in dsFvs, the chainshave been mutated to introduce a disulfide bond to stabilize theassociation of the chains. The term also includes genetically engineeredforms such as chimeric antibodies and heteroconjugate antibodies such asbispecific antibodies. See also, Pierce Catalog and Handbook, 1994-1995(Pierce Chemical Co., Rockford, Ill.); Kuby, Immunology, 3^(rd) Ed.,W.H. Freeman & Co., New York, 1997.

Antibody fragments include, but are not limited to, the following: (1)Fab, the fragment which contains a monovalent antigen-binding fragmentof an antibody molecule produced by digestion of whole antibody with theenzyme papain to yield an intact light chain and a portion of one heavychain; (2) Fab′, the fragment of an antibody molecule obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule; (3) (Fab′)₂, the fragment of theantibody obtained by treating whole antibody with the enzyme pepsinwithout subsequent reduction; (4) F(ab′)₂, a dimer of two Fab′ fragmentsheld together by two disulfide bonds; (5) Fv, a genetically engineeredfragment containing the variable region of the light chain and thevariable region of the heavy chain expressed as two chains; and (6)single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain, the variable regionof the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Antigen binding fragments of an antibody can be produced by themodification of whole antibodies or those synthesized de novo usingrecombinant DNA methodologies. In some examples, the term antibodyincludes the amino acid sequences of one or more of the CDRs from theantibody grafted onto a scaffold.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (κ). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE. The disclosedantibodies can be class switched.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In severalembodiments, the heavy and the light chain variable domains combine tospecifically bind the antigen. In additional embodiments, only the heavychain variable domain is required. For example, naturally occurringcamelid antibodies consisting of a heavy chain only are functional andstable in the absence of light chain (see, e.g., Hamers-Casterman etal., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol.,3:733-736, 1996). Light and heavy chain variable domains contain a“framework” region interrupted by three hypervariable regions, alsocalled “complementarity-determining regions” or “CDRs” (see, e.g., Kabatet al., Sequences of Proteins of Immunological Interest, U.S. Departmentof Health and Human Services, 1991). The sequences of the frameworkregions of different light or heavy chains are relatively conservedwithin a species. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for antigen binding. The sequences ofthe framework regions of different light or heavy chains are relativelyconserved within a species. The framework region of an antibody, that isthe combined framework regions of the constituent light and heavychains, serves to position and align the CDRs in three-dimensionalspace.

The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3(from the N-terminus to C-terminus), and are also typically identifiedby the chain in which the particular CDR is located. Thus, a V_(H) CDR3is located in the variable domain of the heavy chain of the antibody inwhich it is found, whereas a V_(L) CDR1 is the CDR1 from the variabledomain of the light chain of the antibody in which it is found. Lightchain CDRs can also be referred to as CDR L1, CDR L2 and CDR L3, orLCDR1, LCDR2 and LCDR3. Heavy chain CDRs can be referred to as CDR H1,CDR H2 and CDR H3, or HCDR1, HCDR2 and HCDR3.

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an antibody fragment, suchas Fv, scFv, dsFv or Fab. References to “V_(L)” or “VL” refer to thevariable region of an immunoglobulin light chain, including that of anFv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. These fused cells and their progeny are termed“hybridomas”. In some embodiments, monoclonal antibodies can behumanized monoclonal antibodies. In some embodiments, monoclonalantibodies can be chimeric antibodies. In some examples monoclonalantibodies are isolated from a subject. The amino acid sequences of suchisolated monoclonal antibodies can be determined.

A “humanised” antibody is an antibody including a human framework regionand one or more CDRs from anon-human (such as a chimpanzee, mouse, rat,or synthetic) immunoglobulin. The non-human antibody providing the CDRsis termed a “donor”, and the human antibody providing the framework istermed an “acceptor”. In one embodiment, all the CDRs are from the donorantibody in a humanised antibody. Constant regions need not be present,but if they are, they must be substantially identical to human antibodyconstant regions, such as at least about 85-90%, such as about 95% ormore identical. Hence, all parts of a humanised antibody, exceptpossibly the CDRs, are substantially identical to corresponding parts ofnatural human antibody sequences. A “humanised antibody” can include ahumanised light chain and a humanised heavy chain. A humanised antibodybinds to the same antigen as the donor antibody that provides the CDRs.The acceptor framework of a humanised antibody may have a limited numberof substitutions by amino acids taken from the donor framework.Humanised or other monoclonal antibodies can have additionalconservative amino acid substitutions which have substantially no effecton antigen binding or other immunoglobulin functions. Humanisedimmunoglobulins can be constructed by means of genetic engineering (forexample, see U.S. Pat. No. 5,585,089). Preferably, the antibodies of thepresent invention are humanised.

A “chimeric” antibody is an antibody which includes sequences from twodifferent antibodies, which typically are of different species. Forexample, a chimeric antibody may comprise heavy and light chain variableregions derived from a first species and heavy and light chain constantregions derived from a second species. The variable and constant regionsof the light chain may be derived from a first species while thevariable region of the heavy chain may be derived from the first speciesand the constant region of the heavy chain is derived from a secondspecies.

A “neutralizing antibody” is an antibody which reduces effect of avirus, bacteria or tumour for example, by binding to a specific antigenon the virus, bacteria or tumour. In some examples, an antibody that isspecific for a ROR1 neutralises the effect of the tumour.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal.

An antigen reacts with the products of specific humoral or cellularimmunity, including those induced by heterologous antigens, such as thedisclosed antigens. “Epitope” or “antigenic determinant” refers to theregion of an antigen to which B and/or T cells respond. In oneembodiment, T cells respond to the epitope, when the epitope ispresented in conjunction with an MHC molecule. Epitopes can be formedboth from contiguous amino acids or noncontiguous amino acids juxtaposedby tertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5, about 9, or about 8-10 amino acids in a uniquespatial conformation. Methods of determining spatial conformation ofepitopes include, for example, x-ray crystallography and nuclearmagnetic resonance.

Examples of antigens include, but are not limited to, peptides, lipids,polysaccharides, and nucleic acids containing antigenic determinants,such as those recognized by an immune cell. Antigens can includepeptides derived from a pathogen of interest or from a cancerous cell.Exemplary pathogens include bacteria, fungi, viruses and parasites. Insome embodiments, an antigen is derived from a cancerous cell such as ahaematological cancerous cell (chronic lymphocytic leukaemia—CLL, acutelymphoblastic leukaemia, mantle cell lymphoma) or a solid malignancy(breast, pancreatic, melanoma). In some embodiments, the antigen is aROR1 polypeptide or antigenic fragment thereof.

A “target epitope” is a specific epitope on an antigen that specificallybinds an antibody of interest, such as a monoclonal antibody. In someexamples, a target epitope includes the amino acid residues that contactthe antibody of interest, such that the target epitope can be selectedby the amino acid residues determined to be in contact with theantibody.

Binding affinity: Affinity of an antibody or antigen binding fragmentthereof for an antigen. In one embodiment, affinity is calculated by amodification of the Scatchard method described by Frankel et al., Mol.Immunol., 16:101-106, 1979. In another embodiment, binding affinity ismeasured by an antigen/antibody dissociation rate. In yet anotherembodiment, a high binding affinity is measured by a competitionradioimmunoassay. In several examples, a high binding affinity is atleast about 1×10⁻⁸ M. In other embodiments, a high binding affinity isat least about 1.5×10⁻⁸, at least about 2.0×10⁻⁸, at least about2.5×10⁻⁸, at least about 3.0×10⁻⁸, at least about 3.5×10⁻⁸, at leastabout 4.0×10⁻⁸, at least about 4.5×10⁻⁸, at least about 5.0×10⁻⁸ M, orat least about 1×10⁻⁹ M.

Clonal variant: Any sequence, which differs by one or more nucleotidesor amino acids, in presence of V region with identical mutationscompared to the germline, identical VDJ or VJ gene usage, and identicalD and J length. The “germline” sequence is intended to be the sequencecoding for the antibody/immunoglobulin (or of any fragment thereof)deprived of mutations, for example somatic mutations. The percentage ofhomology represents an indication of the mutational events which anytype of heavy chain portion undergoes after contact with an antigen.

Conjugate: A complex of two molecules linked together, for example,linked together by a covalent bond. In one embodiment, an antibody islinked to an effector molecule; for example, an antibody thatspecifically binds to a ROR1 polypeptide, covalently linked to aneffector molecule or to a toxin. The linkage can be by chemical orrecombinant means. In one embodiment, the linkage is chemical, wherein areaction between the antibody moiety and the effector molecule hasproduced a covalent bond formed between the two molecules to form onemolecule. A peptide linker (short peptide sequence) can optionally beincluded between the antibody and the effector molecule. Becauseconjugates can be prepared from two molecules with separatefunctionalities, such as an antibody and an effector molecule, they arealso sometimes referred to as “chimeric molecules.” In one embodiment,an antibody linked to an effector molecule is further joined to a lipidor other molecule to a protein or peptide to increase its half-life inthe body.

Contacting: Placement in direct physical association; includes both insolid and liquid form, which can take place either in vivo or in vitro.Contacting includes contact between one molecule and another molecule,for example the amino acid on the surface of one polypeptide, such as anantigen, that contacts another polypeptide, such as an antibody.Contacting can also include contacting a cell for example by placing anantibody in direct physical association with a cell.

Control: A reference standard. In some embodiments, the control is asample obtained from a healthy patient. In other embodiments, thecontrol is a tissue sample obtained from a patient diagnosed with cancerthat serves as a positive control. In still other embodiments, thecontrol is a historical control or standard reference value or range ofvalues (such as a previously tested control sample, such as a group ofinfected patients with known prognosis or outcome, or group of samplesthat represent baseline or normal values).

A difference between a test sample and a control can be an increase orconversely a decrease. The difference can be a qualitative difference ora quantitative difference, for example a statistically significantdifference. In some examples, a difference is an increase or decrease,relative to a control, of at least about 5%, such as at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, at least about 500%, or greater than 500%.

Detectable marker: A detectable molecule (also known as a label) that isconjugated directly or indirectly to a second molecule, such as anantibody, to facilitate detection of the second molecule. For example,the detectable marker can be capable of detection by ELISA,spectrophotometry, flow cytometry, microscopy or diagnostic imagingtechniques (such as CT scans, MRIs, ultrasound, fiberoptic examination,and laparoscopic examination). Non-limiting examples of detectablemarkers include fluorophores, fluorescent proteins, chemiluminescentagents, enzymatic linkages, radioactive isotopes and heavy metals orcompounds (for example super paramagnetic iron oxide nanocrystals fordetection by MRI). In one example, a “labeled antibody” refers toincorporation of another molecule in the antibody. For example, thelabel is a detectable marker, such as the incorporation of aradiolabeled amino acid or attachment to a polypeptide of biotinylmoieties that can be detected by marked avidin (for example,streptavidin containing a fluorescent marker or enzymatic activity thatcan be detected by optical or colorimetric methods). Various methods oflabeling polypeptides and glycoproteins are known in the art and may beused. Examples of labels for polypeptides include, but are not limitedto, the following: radioisotopes or radionuclides (such as ³⁵S or ¹³¹I),fluorescent labels (such as fluorescein isothiocyanate (FITC),rhodamine, lanthanide phosphors), enzymatic labels (such as horseradishperoxidase, beta-galactosidase, luciferase, alkaline phosphatase),chemiluminescent markers, biotinyl groups, predetermined polypeptideepitopes recognized by a secondary reporter (such as a leucine zipperpair sequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags), or magnetic agents, such as gadolinium chelates.In some embodiments, labels are attached by spacer arms of variouslengths to reduce potential steric hindrance. Methods for usingdetectable markers and guidance in the choice of detectable markersappropriate for various purposes are discussed for example in Sambrooket al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y., 1989) and Ausubel et al. (In Current Protocols in MolecularBiology, John Wiley & Sons, New York, 1998). In particular embodimentsof the invention, the antibody or fragment thereof can be labelled witha detectable marker.

Detecting: To identify the existence, presence, or fact of something.General methods of detecting are known to the skilled artisan (see, forexample, U.S. Pat. No. 7,635,476) and may be supplemented with theprotocols and reagents disclosed herein. For example, included hereinare methods of detecting a cell that expresses a ROR1 polypeptide in asubject.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide. In some examples a disclosedantibody specifically binds to an epitope on the surface of ROR1.

Framework Region: Amino acid sequences interposed between CDRs. The termincludes variable light and variable heavy framework regions. Theframework regions serve to hold the CDRs in an appropriate orientationfor antigen binding.

Fc polypeptide: The polypeptide comprising the constant region of anantibody excluding the first constant region immunoglobulin domain. Fcregion generally refers to the last two constant region immunoglobulindomains of IgA, IgD, and IgG, and the last three constant regionimmunoglobulin domains of IgE and IgM. An Fc region may also includepart or all of the flexible hinge N-terminal to these domains. For IgAand IgM, an Fc region may or may not comprise the tailpiece, and may ormay not be bound by the J chain. For IgG, the Fc region comprisesimmunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the lowerpart of the hinge between Cgamma1 (Cγ1) and Cγ2. Although the boundariesof the Fc region may vary, the human IgG heavy chain Fc region isusually defined to comprise residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index.For IgA, the Fc region comprises immunoglobulin domains Calpha2 andCalpha3 (Cα2 and Cα3) and the lower part of the hinge between Calpha1(Cα1) and Cα2. Encompassed within the definition of the Fc region arefunctionally equivalent analogs and variants of the Fc region. Afunctionally equivalent analog of the Fc region may be a variant Fcregion, comprising one or more amino acid modifications relative to thewild-type or naturally existing Fc region. Variant Fc regions willpossess at least 50% homology with a naturally existing Fc region, suchas about 80%, and about 90%, or at least about 95% homology.Functionally equivalent analogs of the Fc region may comprise one ormore amino acid residues added to or deleted from the N- or C-termini ofthe protein, such as no more than 30 or no more than 10 additions and/ordeletions. Functionally equivalent analogs of the Fc region include Fcregions operably linked to a fusion partner. Functionally equivalentanalogs of the Fc region must comprise the majority of all of the Igdomains that compose Fc region as defined above; for example IgG and IgAFc regions as defined herein must comprise the majority of the sequenceencoding CH₂ and the majority of the sequence encoding CH₃. Thus, theCH₂ domain on its own, or the CH₃ domain on its own, are not consideredFc region. The Fc region may refer to this region in isolation, or thisregion in the context of an Fc fusion polypeptide.

Host cells: Cells in which a vector can be propagated and its DNAexpressed, for example a disclosed antibody can be expressed in a hostcell. The cell may be prokaryotic or eukaryotic. The term also includesany progeny of the subject host cell. It is understood that all progenymay not be identical to the parental cell since there may be mutationsthat occur during replication. However, such progeny are included whenthe term “host cell” is used.

Immune complex: The binding of antibody to a soluble antigen forms animmune complex. The formation of an immune complex can be detectedthrough conventional methods known to the skilled artisan, for instanceimmunohistochemistry, immunoprecipitation, flow cytometry,immunofluorescence microscopy, ELISA, immunoblotting (for example,Western blot), magnetic resonance imaging, CT scans, X-ray and affinitychromatography. Immunological binding properties of selected antibodiesmay be quantified using methods well known in the art.

Immunologically reactive conditions: Includes reference to conditionswhich allow an antibody raised against a particular epitope to bind tothat epitope to a detectably greater degree than, and/or to thesubstantial exclusion of, binding to substantially all other epitopes.Immunologically reactive conditions are dependent upon the format of theantibody binding reaction and typically are those utilized inimmunoassay protocols or those conditions encountered in vivo. SeeHarlow & Lane, supra, for a description of immunoassay formats andconditions. The immunologically reactive conditions employed in themethods are “physiological conditions” which include reference toconditions (e.g., temperature, osmolarity, pH) that are typical inside aliving mammal or a mammalian cell. While it is recognized that someorgans are subject to extreme conditions, the intra-organismal andintracellular environment normally lies around pH 7 (e.g., from pH 6.0to pH 8.0, more typically pH 6.5 to 7.5), contains water as thepredominant solvent, and exists at a temperature above 0° C. and below50° C. Osmolarity is within the range that is supportive of cellviability and proliferation.

Inhibiting or treating a disease: Inhibiting the full development of adisease or condition, for example, in a subject who is at risk forcancer. “Treatment” refers to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological conditionafter it has begun to develop. The term “ameliorating,” with referenceto a disease or pathological condition, refers to any observablebeneficial effect of the treatment. The beneficial effect can beevidenced, for example, by a delayed onset of clinical symptoms of thedisease in a susceptible subject, a reduction in severity of some or allclinical symptoms of the disease, a slower progression of the disease, areduction in size of the tumour/cancer, an improvement in the overallhealth or well-being of the subject, or by other parameters well knownin the art that are specific to the particular disease. A “prophylactic”treatment is a treatment administered to a subject who does not exhibitsigns of a disease or exhibits only early signs for the purpose ofdecreasing the risk of developing pathology.

Isolated: An “isolated” biological component (such as a cell, forexample a B-cell, a nucleic acid, peptide, protein, heavy chain domainor antibody) has been substantially separated, produced apart from, orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, such as, otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins which have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides, and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids. In some examples an antibody, such as an antibodyspecific for a ROR1 polypeptide can be isolated, for example isolatedfrom a subject with a tumour expressing ROR1.

K_(d): The dissociation constant for a given interaction, such as apolypeptide ligand interaction or an antibody antigen interaction. Forexample, for the bimolecular interaction of an antibody (such as any ofthe antibodies disclosed herein) and an antigen (such as a ROR1polypeptide) it is the concentration of the individual components of thebimolecular interaction divided by the concentration of the complex.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule, such as an antibody or a protein, tofacilitate detection of that molecule. Specific, non-limiting examplesof labels include fluorescent tags, enzymatic linkages, and radioactiveisotopes. In some examples, a disclosed antibody is labeled.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Recombinant nucleic acid” refers to a nucleic acid having nucleotidesequences that are not naturally joined together. This includes nucleicacid vectors comprising an amplified or assembled nucleic acid which canbe used to transform a suitable host cell. A host cell that comprisesthe recombinant nucleic acid is referred to as a “recombinant hostcell.” The gene is then expressed in the recombinant host cell toproduce, e.g., a “recombinant polypeptide.” A recombinant nucleic acidmay serve a non-coding function (e.g., promoter, origin of replication,ribosome-binding site, etc.) as well.

A first sequence is an “antisense” with respect to a second sequence ifa polynucleotide whose sequence is the first sequence specificallyhybridizes with a polynucleotide whose sequence is the second sequence.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

For sequence comparison of nucleic acid sequences, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443, 1970, by the search for similarity method of Pearson & Lipman,Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Current Protocols in Molecular Biology (Ausubelet al., eds 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360, 1987. The method used is similar to the methoddescribed by Higgins & Sharp, CABIOS 5:151-153, 1989. Using PILEUP, areference sequence is compared to other test sequences to determine thepercent sequence identity relationship using the following parameters:default gap weight (3.00), default gap length weight (0.10), andweighted end gaps. PILEUP can be obtained from the GCG sequence analysissoftware package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res.12:387-395, 1984.

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and the BLAST2.0 algorithm, which are described in Altschul et al., J Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) usesas defaults a word length (W) of 11, alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTPprogram (for amino acid sequences) uses as defaults a word length (W) of3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). Anoligonucleotide is a linear polynucleotide sequence of up to about 100nucleotide bases in length.

ClustalW is a program that aligns three or more sequences in acomputationally efficient manner. Aligning multiple sequences highlightsareas of similarity which may be associated with specific features thathave been more highly conserved than other regions. Thus, this programcan classify sequences for phylogenetic analysis, which aims to modelthe substitutions that have occurred over evolution and derive theevolutionary relationships between sequences. The ClustalW multiplesequence alignment web form is available on the internet from EMBL-EBI(ebi.ac.uk/Tools/msa/clustalw2/), see also Larkin et al., Bioinformatics2007 23(21): 2947-2948.

A polynucleotide or nucleic acid sequence refers to a polymeric form ofnucleotide at least 10 bases in length. A recombinant polynucleotideincludes a polynucleotide that is not immediately contiguous with bothof the coding sequences with which it is immediately contiguous (one onthe 5′ end and one on the 3′ end) in the naturally occurring genome ofthe organism from which it is derived. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector; into anautonomously replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA) independent of other sequences. The nucleotides can beribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. The term includes single- and double-stranded forms of DNA.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995,describes compositions and formulations suitable for pharmaceuticaldelivery of the antibodies herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids, which include, but are not limitedto, water, physiological saline, balanced salt solutions, aqueousdextrose, glycerol or the like as a vehicle. For solid compositions(e.g., powder, pill, tablet, or capsule forms), conventional non-toxicsolid carriers can include, for example, pharmaceutical grades ofmannitol, lactose, starch, or magnesium stearate. In addition tobiologically neutral carriers, pharmaceutical compositions to beadministered can contain minor amounts of non-toxic auxiliarysubstances, such as wetting or emulsifying agents, preservatives, and pHbuffering agents and the like, for example sodium acetate or sorbitanmonolaurate.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell. In some examples a pharmaceuticalagent includes one or more of the disclosed antibodies.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). In one embodiment, the polypeptide is a ROR1polypeptide. In one embodiment, the polypeptide is a disclosed antibodyor a fragment thereof. A “residue” refers to an amino acid or amino acidmimetic incorporated in a polypeptide by an amide bond or amide bondmimetic. A polypeptide has an amino terminal (N-terminal) end and acarboxy terminal end. Conservative amino acid substitution tablesproviding functionally similar amino acids are well known to one ofordinary skill in the art. The following six groups are examples ofamino acids that are considered to be conservative substitutions for oneanother:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein (such as an antibody)is more enriched than the peptide or protein is in its naturalenvironment within a cell. In one embodiment, a preparation is purifiedsuch that the protein or peptide represents at least 50% of the totalpeptide or protein content of the preparation.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a polypeptide will possess a relatively highdegree of sequence identity when aligned using standard methods.

Methods of alignment of polypeptide sequences for comparison are wellknown in the art. Various programs and alignment algorithms may be usedas described above. Altschul et al., Nature Genet. 6:119, 1994, presentsa detailed consideration of sequence alignment methods and homologycalculations. The NCBI Basic Local Alignment Search Tool (BLAST)(Altschul et al., J. Mol. Biol. 215:403, 1990) is available from severalsources, including the National Center for Biotechnology Information(NCBI, Bethesda, Md.) and on the internet (along with a description ofhow to determine sequence identity using this program).

Homologs and variants of a V_(L) or a V_(H) of an antibody thatspecifically binds a polypeptide are typically characterized bypossession of at least about 75%, for example at least about 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identitycounted over the full length alignment with the amino acid sequence ofinterest. Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% sequence identity. When less than theentire sequence is being compared for sequence identity, homologs andvariants will typically possess at least 80% sequence identity overshort windows of 10-20 amino acids, and may possess sequence identitiesof at least 85% or at least 90% or 95% depending on their similarity tothe reference sequence. One of skill in the art will appreciate thatthese sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided.

Nucleic acids that “selectively hybridise” or “selectively bind” do sounder moderately or highly stringent conditions that excludesnon-related nucleotide sequences. In nucleic acid hybridisationreactions, the conditions used to achieve a particular level ofstringency will vary, depending on the nature of the nucleic acids beinghybridised. For example, the length, degree of complementarity,nucleotide sequence composition (for example, GC v. AT content), andnucleic acid type (for example, RNA versus DNA) of the hybridisingregions of the nucleic acids can be considered in selectinghybridisation conditions. An additional consideration is whether one ofthe nucleic acids is immobilised, for example, on a filter.

A specific example of progressively higher stringency conditions is asfollows: 2×SSC/0.1% SDS at about room temperature (hybridizationconditions); 0.2×SSC/0.1% SDS at about room temperature (low stringencyconditions); 0.2×SSC/0.1% SDS at about 42° C. (moderate stringencyconditions); and 0.1×SSC at about 68° C. (high stringency conditions).One of skill in the art can readily determine variations on theseconditions (e.g., Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989). Washing can be carried out using only one ofthese conditions, e.g., high stringency conditions, or each of theconditions can be used, e.g., for 10-15 minutes each, in the orderlisted above, repeating any or all of the steps listed. However, asmentioned above, optimal conditions will vary, depending on theparticular hybridization reaction involved, and can be determinedempirically.

Specifically bind: When referring to an antibody, refers to a bindingreaction which determines the presence of a target protein, peptide, orpolysaccharide in the presence of a heterogeneous population of proteinsand other biologics. Thus, under designated conditions, an antibodybinds preferentially to a particular target protein, peptide orpolysaccharide (such as an antigen present on the surface of a tumour,for example ROR1) and do not bind in a significant amount to otherproteins or polysaccharides present in the sample or subject. Specificbinding can be determined by methods known in the art. With reference toan antibody antigen complex, specific binding of the antigen andantibody has a K_(d) of less than about 10⁻⁷ Molar, such as less thanabout 10⁻⁷ Molar, 10⁻⁸ Molar, 10⁻⁹, or even less than about 10⁻¹⁰ Molar.

Therapeutic agent: Used in a generic sense, it includes treating agents,prophylactic agents, and replacement agents.

Therapeutically effective amount or effective amount: A quantity of aspecific substance, such as a disclosed antibody, sufficient to achievea desired effect in a subject being treated. For instance, this can bethe amount necessary to inhibit tumour growth. In several embodiments, atherapeutically effective amount is the amount necessary to reduce asymptom of the disease. When administered to a subject, a dosage willgenerally be used that will achieve target tissue concentrations thathas been shown to achieve a desired in vitro effect.

Vector: A nucleic acid molecule may be introduced into a host cell by avector, thereby producing a transformed host cell. A vector may includenucleic acid sequences that permit it to replicate in a host cell, suchas an origin of replication. A vector may also include one or moreselectable marker genes and other genetic elements known in the art.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. The term“comprises” means “includes.” All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

Monoclonal Antibodies that Specifically Bind to ROR1

Clinically and diagnostically useful rat-derived monoclonal antibodies(MAb) that specifically bind ROR1 are disclosed herein.

In some embodiments the monoclonal antibodies specifically bind a ROR1polypeptide with an equilibrium constant (K_(d)) of about 6×10⁻⁹ M orless. In some embodiments, the monoclonal antibodies specifically bind aROR1 polypeptide with a K_(d) of about 1.6×10⁻⁹ M or less, about 2×10⁻⁹M or less, about 3×10⁻⁹ M or less, about 4×10⁻⁹ M or less or about5×10⁻⁹ M or less.

The MAb can be of any isotype. The MAb can be, for example, an IgM or anIgG antibody, such as IgG₁ or an IgG₂. The class of an antibody thatspecifically binds ROR1 can be switched with another. In one aspect, anucleic acid molecule encoding V_(L) or V_(H) is isolated using methodswell-known in the art, such that it does not include any nucleic acidsequences encoding the constant region of the light or heavy chain,respectively. The nucleic acid molecule encoding V_(L) or V_(H) is thenoperatively linked to a nucleic acid sequence encoding a CL or CH from adifferent class of immunoglobulin molecule. This can be achieved using avector or nucleic acid molecule that comprises a CL or CH chain, asknown in the art. For example, an antibody that specifically binds ROR1that was originally IgM may be class switched to an IgG. Class switchingcan be used to convert one IgG subclass to another, such as from IgG₁ toIgG₂.

The monoclonal antibodies disclosed herein can be rat antibodies, andcan include a rat framework region. In some preferred embodiments, theantibodies are humanised, and thus include one or more human frameworkregions. In some embodiments, the MAbs disclosed herein are chimericantibodies. In some embodiments, the MAbs include rat and human regions.

The monoclonal antibody can specifically bind a ROR1 polypeptide.Preferably, the monoclonal antibody can specifically bind a human ROR1polypeptide. The antibody preferably comprises a heavy chain and a lightchain and preferably each VH and VL is composed of three CDRs and fourFWRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4 as described above.

In a first embodiment, the isolated monoclonal antibody comprises alight chain variable domain and a heavy chain variable domain whereinthe light chain variable domain comprises a light chain complementaritydetermining region (LCDR)1, an LCDR2 and an LCDR3, wherein LCDR1comprises an amino acid sequence set forth in SEQ ID NO: 30, SEQ ID NO:24, SEQ ID NO: 9, SEQ ID NO: 37, SEQ ID NO: 44, SEQ ID NO: 50, SEQ IDNO: 56, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 2, SEQ ID NO: 16, orSEQ ID NO: 22; LCDR2 comprises an amino acid sequence set forth in SEQID NO: 32, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 39, SEQ ID NO: 46,SEQ ID NO: 52, SEQ ID NO: 58, SEQ ID NO: 4 or SEQ ID NO: 18; and LCDR3comprises an amino acid sequence set forth in SEQ ID NO: 34, SEQ ID NO:28, SEQ ID NO: 13, SEQ ID NO: 41, SEQ ID NO: 48, SEQ ID NO: 54, SEQ IDNO: 60, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 6, orSEQ ID NO: 20; and wherein the heavy chain variable domain comprises aheavy chain complementarity determining region (HCDR)₁, an HCDR2 and anHCDR3, wherein HCDR1 comprises an amino acid sequence set forth in SEQID NO: 121, SEQ ID NO: 115, SEQ ID NO: 96, SEQ ID NO: 127, SEQ ID NO:134, SEQ ID NO: 140, SEQ ID NO: 146, SEQ ID NO: 152, SEQ ID NO: 158, SEQID NO: 163, SEQ ID NO: 89, SEQ ID NO: 103 or SEQ ID NO: 110; HCDR2comprises an amino acid sequence set forth in SEQ ID NO: 123, SEQ ID NO:117, SEQ ID NO: 98, SEQ ID NO: 129, SEQ ID NO: 136, SEQ ID NO: 142, SEQID NO: 148, SEQ ID NO: 154, SEQ ID NO: 160, SEQ ID NO: 165, SEQ ID NO:91 or SEQ ID NO: 105; and HCDR3 comprises an amino acid sequences setforth in SEQ ID NO: 248, SEQ ID NO: 252, SEQ ID NO: 100, SEQ ID NO: 131,SEQ ID NO: 138, SEQ ID NO: 144, SEQ ID NO: 150, SEQ ID NO: 156, SEQ IDNO: 162, SEQ ID NO: 167, SEQ ID NO: 93, SEQ ID NO: 107 or SEQ ID NO:113, wherein the sequence of each complementarity determining region maydiffer from the given sequence at up to two amino acid positions, andwherein the monoclonal antibody specifically binds to a ROR1polypeptide.

The isolated monoclonal antibody may comprises a light chain variabledomain and a heavy chain variable domain wherein the light chainvariable domain comprises a light chain complementarity determiningregion (LCDR)1, an LCDR2 and an LCDR3, wherein LCDR1 comprises an aminoacid sequence set forth in SEQ ID NO: 30, SEQ ID NO: 24, SEQ ID NO: 9,SEQ ID NO: 37, SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 56, SEQ ID NO:67 or SEQ ID NO: 71; LCDR2 comprises an amino acid sequence set forth inSEQ ID NO: 32, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 39, SEQ ID NO:46, SEQ ID NO: 52 or SEQ ID NO: 58; and LCDR3 comprises an amino acidsequence set forth in SEQ ID NO: 34, SEQ ID NO: 28, SEQ ID NO: 13, SEQID NO: 41, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 65,SEQ ID NO: 68 or SEQ ID NO: 74; and wherein the heavy chain variabledomain comprises a heavy chain complementarity determining region(HCDR)1, an HCDR2 and an HCDR3, wherein HCDR1 comprises an amino acidsequence set forth in SEQ ID NO: 121, SEQ ID NO: 115, SEQ ID NO: 96, SEQID NO: 127, SEQ ID NO: 134, SEQ ID NO: 140, SEQ ID NO: 146, SEQ ID NO:152, SEQ ID NO: 158 or SEQ ID NO: 163; HCDR2 comprises an amino acidsequence set forth in SEQ ID NO: 123, SEQ ID NO: 117, SEQ ID NO: 98, SEQID NO: 129, SEQ ID NO: 136, SEQ ID NO: 142, SEQ ID NO: 148, SEQ ID NO:154, SEQ ID NO: 160 or SEQ ID NO: 165; and HCDR3 comprises an amino acidsequences set forth in SEQ ID NO: 248, SEQ ID NO: 252, SEQ ID NO: 100,SEQ ID NO: 131, SEQ ID NO: 138, SEQ ID NO: 144, SEQ ID NO: 150, SEQ IDNO: 156, SEQ ID NO: 162 or SEQ ID NO: 167, wherein the sequence of eachcomplementarity determining region may differ from the given sequence atup to two amino acid positions, and wherein the monoclonal antibodyspecifically binds to a ROR1 polypeptide.

Preferably the isolated monoclonal antibody comprises a light chainvariable domain, wherein the light chain variable domain comprises alight chain complementarity determining region (LCDR)1, an LCDR2 and anLCDR3, wherein

-   -   (a) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 30, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 32, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 34;    -   (b) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 24, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 26, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 28;    -   (c) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 9, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 11, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 13;    -   (d) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 37, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 39, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 41;    -   (e) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 44, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 46, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 48;    -   (f) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 50, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 52, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 54;    -   (g) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 56, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 58, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 60;    -   (h) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 44, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 46, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 65;    -   (i) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 67, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 58, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 68;    -   (j) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 71, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 26, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 74;    -   (k) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 2, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 4, and the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 6;    -   (l) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 16, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 18, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 20; or    -   (m) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 22, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 18, and the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 20;    -   wherein the sequence of each complementarity determining region        may differ from the given sequence at up to two amino acid        positions, and    -   wherein the monoclonal antibody specifically binds to a ROR1        polypeptide.

Preferably the isolated monoclonal antibody comprises a heavy chainvariable domain, wherein the heavy chain variable domain comprises aheavy chain complementarity determining region (HCDR)1, an HCDR2 and anHCDR3, wherein

-   -   (a) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 121, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 123, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 248;    -   (b) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 115, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 117, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 252;    -   (c) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 96, the HCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 98, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 100;    -   (d) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 127, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 129, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 131;    -   (e) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 134, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 136, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 138;    -   (f) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 140, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 142, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 144;    -   (g) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 146, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 148, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 150;    -   (h) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 152, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 154, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 156;    -   (i) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 158, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 160, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 162;    -   (j) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 163, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 165, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 167;    -   (k) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 89, the HCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 91, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 93;    -   (l) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 103, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 105, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 107; or    -   (m) the HCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 110, the HCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 105, and the HCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 113;    -   wherein the sequence of each complementarity determining region        may differ from the given sequence at up to two amino acid        positions, and    -   wherein the monoclonal antibody specifically binds to a ROR1        polypeptide.

In a particularly preferred embodiment, the isolated monoclonal antibodycomprises a light chain variable domain and a heavy chain variabledomain, wherein the light chain variable domain comprises a light chaincomplementarity determining region (LCDR)1, an LCDR2 and an LCDR3 andthe heavy chain variable domain comprises a heavy chain complementaritydetermining region (HCDR)1, an HCDR2 and an HCDR3, wherein

-   -   (a) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 30, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 32, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 34, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 121, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 123, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 248;    -   (b) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 24, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 26, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 28, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 115, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 117, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 252;    -   (c) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 9, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 11, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 13, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 96, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 98, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 100;    -   (d) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 37, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 39, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 41, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 127, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 129, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 131;    -   (e) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 44, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 46, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 48, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 134, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 136, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 138;    -   (f) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 50, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 52, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 54, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 140, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 142, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 144;    -   (g) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 56, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 58, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 60, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 146, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 148, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 150;    -   (h) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 44, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 46, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 65, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 152, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 154, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 156;    -   (i) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 67, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 58, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 68, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 158, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 160, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 162;    -   (j) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 71, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 26, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 74, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 163, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 165, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 167;    -   (k) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 2, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 4, the LCDR3 comprises the amino acid sequence set        forth as SEQ ID NO: 6, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 89, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 91, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 93;    -   (l) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 16, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 18, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 20, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 103, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO:105, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 107;        or    -   (m) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 22, the LCDR2 comprises the amino acid sequence set forth        as SEQ ID NO: 18, the LCDR3 comprises the amino acid sequence        set forth as SEQ ID NO: 20, the HCDR1 comprises the amino acid        sequence set forth as SEQ ID NO: 110, the HCDR2 comprises the        amino acid sequence set forth as SEQ ID NO: 105, and the HCDR3        comprises the amino acid sequence set forth as SEQ ID NO: 113;    -   wherein the sequence of each complementarity determining region        may differ from the given sequence at up to two amino acid        positions, and    -   wherein the monoclonal antibody specifically binds to a ROR1        polypeptide.

The isolated monoclonal antibody may comprise a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises a light chain complementarity determiningregion (LCDR)1, an LCDR2 and an LCDR3 and the heavy chain variabledomain comprises a heavy chain complementarity determining region(HCDR)1, an HCDR2 and an HCDR3, wherein the LCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 30, the LCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 32, the LCDR3 comprises the aminoacid sequence set forth as SEQ ID NO: 34, the HCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 121, the HCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 123, and the HCDR3 comprises theamino acid sequence set forth as SEQ ID NO: 248; wherein the sequence ofeach complementarity determining region may differ from the givensequence at up to two amino acid positions, and wherein the monoclonalantibody specifically binds to a ROR1 polypeptide.

The isolated monoclonal antibody may comprise a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises a light chain complementarity determiningregion (LCDR)1, an LCDR2 and an LCDR3 and the heavy chain variabledomain comprises a heavy chain complementarity determining region(HCDR)1, an HCDR2 and an HCDR3, wherein the LCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 24, the LCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 26, the LCDR3 comprises the aminoacid sequence set forth as SEQ ID NO: 28, the HCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 115, the HCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 117, and the HCDR3 comprises theamino acid sequence set forth as SEQ ID NO: 252; wherein the sequence ofeach complementarity determining region may differ from the givensequence at up to two amino acid positions, and wherein the monoclonalantibody specifically binds to a ROR1 polypeptide.

The isolated monoclonal antibody may comprise a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises a light chain complementarity determiningregion (LCDR)1, an LCDR2 and an LCDR3 and the heavy chain variabledomain comprises a heavy chain complementarity determining region(HCDR)1, an HCDR2 and an HCDR3, wherein the LCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 9, the LCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 11, the LCDR3 comprises the aminoacid sequence set forth as SEQ ID NO: 13, the HCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 96, the HCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 98, and the HCDR3 comprises theamino acid sequence set forth as SEQ ID NO: 100; wherein the sequence ofeach complementarity determining region may differ from the givensequence at up to two amino acid positions, and wherein the monoclonalantibody specifically binds to a ROR1 polypeptide.

The isolated monoclonal antibody may comprise a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises a light chain complementarity determiningregion (LCDR)1, an LCDR2 and an LCDR3 and the heavy chain variabledomain comprises a heavy chain complementarity determining region(HCDR)1, an HCDR2 and an HCDR3, wherein the LCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 37, the LCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 39, the LCDR3 comprises the aminoacid sequence set forth as SEQ ID NO: 41, the HCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 127, the HCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 129, and the HCDR3 comprises theamino acid sequence set forth as SEQ ID NO: 131; wherein the sequence ofeach complementarity determining region may differ from the givensequence at up to two amino acid positions, and wherein the monoclonalantibody specifically binds to a ROR1 polypeptide.

The isolated monoclonal antibody may comprise a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises a light chain complementarity determiningregion (LCDR)1, an LCDR2 and an LCDR3 and the heavy chain variabledomain comprises a heavy chain complementarity determining region(HCDR)1, an HCDR2 and an HCDR3, wherein the LCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 44, the LCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 46, the LCDR3 comprises the aminoacid sequence set forth as SEQ ID NO: 48, the HCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 134, the HCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 136, and the HCDR3 comprises theamino acid sequence set forth as SEQ ID NO: 138; wherein the sequence ofeach complementarity determining region may differ from the givensequence at up to two amino acid positions, and wherein the monoclonalantibody specifically binds to a ROR1 polypeptide.

The isolated monoclonal antibody may comprise a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises a light chain complementarity determiningregion (LCDR)1, an LCDR2 and an LCDR3 and the heavy chain variabledomain comprises a heavy chain complementarity determining region(HCDR)1, an HCDR2 and an HCDR3, wherein the LCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 50, the LCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 52, the LCDR3 comprises the aminoacid sequence set forth as SEQ ID NO: 54, the HCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 140, the HCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 142, and the HCDR3 comprises theamino acid sequence set forth as SEQ ID NO: 144; wherein the sequence ofeach complementarity determining region may differ from the givensequence at up to two amino acid positions, and wherein the monoclonalantibody specifically binds to a ROR1 polypeptide.

The isolated monoclonal antibody may comprise a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises a light chain complementarity determiningregion (LCDR)1, an LCDR2 and an LCDR3 and the heavy chain variabledomain comprises a heavy chain complementarity determining region(HCDR)1, an HCDR2 and an HCDR3, wherein the LCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 56, the LCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 58, the LCDR3 comprises the aminoacid sequence set forth as SEQ ID NO: 60, the HCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 146, the HCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 148, and the HCDR3 comprises theamino acid sequence set forth as SEQ ID NO: 150; wherein the sequence ofeach complementarity determining region may differ from the givensequence at up to two amino acid positions, and wherein the monoclonalantibody specifically binds to a ROR1 polypeptide.

The isolated monoclonal antibody may comprise a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises a light chain complementarity determiningregion (LCDR)1, an LCDR2 and an LCDR3 and the heavy chain variabledomain comprises a heavy chain complementarity determining region(HCDR)1, an HCDR2 and an HCDR3, wherein the LCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 44, the LCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 46, the LCDR3 comprises the aminoacid sequence set forth as SEQ ID NO: 65, the HCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 152, the HCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 154, and the HCDR3 comprises theamino acid sequence set forth as SEQ ID NO: 156; wherein the sequence ofeach complementarity determining region may differ from the givensequence at up to two amino acid positions, and wherein the monoclonalantibody specifically binds to a ROR1 polypeptide.

The isolated monoclonal antibody may comprise a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises a light chain complementarity determiningregion (LCDR)1, an LCDR2 and an LCDR3 and the heavy chain variabledomain comprises a heavy chain complementarity determining region(HCDR)1, an HCDR2 and an HCDR3, wherein the LCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 67, the LCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 58, the LCDR3 comprises the aminoacid sequence set forth as SEQ ID NO: 68, the HCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 158, the HCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 160, and the HCDR3 comprises theamino acid sequence set forth as SEQ ID NO: 162; wherein the sequence ofeach complementarity determining region may differ from the givensequence at up to two amino acid positions, and wherein the monoclonalantibody specifically binds to a ROR1 polypeptide.

The isolated monoclonal antibody may comprise a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises a light chain complementarity determiningregion (LCDR)1, an LCDR2 and an LCDR3 and the heavy chain variabledomain comprises a heavy chain complementarity determining region(HCDR)1, an HCDR2 and an HCDR3, wherein the LCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 71, the LCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 26, the LCDR3 comprises the aminoacid sequence set forth as SEQ ID NO: 74, the HCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 163, the HCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 165, and the HCDR3 comprises theamino acid sequence set forth as SEQ ID NO: 167; wherein the sequence ofeach complementarity determining region may differ from the givensequence at up to two amino acid positions, and wherein the monoclonalantibody specifically binds to a ROR1 polypeptide.

The isolated monoclonal antibody may comprise a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises a light chain complementarity determiningregion (LCDR)1, an LCDR2 and an LCDR3 and the heavy chain variabledomain comprises a heavy chain complementarity determining region(HCDR)1, an HCDR2 and an HCDR3, wherein the LCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 2, the LCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 4, the LCDR3 comprises the aminoacid sequence set forth as SEQ ID NO: 6, the HCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 89, the HCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 91, and the HCDR3 comprises theamino acid sequence set forth as SEQ ID NO: 93; wherein the sequence ofeach complementarity determining region may differ from the givensequence at up to two amino acid positions, and wherein the monoclonalantibody specifically binds to a ROR1 polypeptide.

The isolated monoclonal antibody may comprise a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises a light chain complementarity determiningregion (LCDR)1, an LCDR2 and an LCDR3 and the heavy chain variabledomain comprises a heavy chain complementarity determining region(HCDR)1, an HCDR2 and an HCDR3, wherein the LCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 16, the LCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 18, the LCDR3 comprises the aminoacid sequence set forth as SEQ ID NO: 20, the HCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 103, the HCDR2 comprises the aminoacid sequence set forth as SEQ ID NO:105, and the HCDR3 comprises theamino acid sequence set forth as SEQ ID NO: 107; wherein the sequence ofeach complementarity determining region may differ from the givensequence at up to two amino acid positions, and wherein the monoclonalantibody specifically binds to a ROR1 polypeptide.

The isolated monoclonal antibody may comprise a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises a light chain complementarity determiningregion (LCDR)1, an LCDR2 and an LCDR3 and the heavy chain variabledomain comprises a heavy chain complementarity determining region(HCDR)1, an HCDR2 and an HCDR3, wherein the LCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 22, the LCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 18, the LCDR3 comprises the aminoacid sequence set forth as SEQ ID NO: 20, the HCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 110, the HCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 105, and the HCDR3 comprises theamino acid sequence set forth as SEQ ID NO: 113; wherein the sequence ofeach complementarity determining region may differ from the givensequence at up to two amino acid positions, and wherein the monoclonalantibody specifically binds to a ROR1 polypeptide.

The isolated monoclonal antibody may comprise a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises a light chain complementarity determiningregion (LCDR)1, an LCDR2 and an LCDR3 and the heavy chain variabledomain comprises a heavy chain complementarity determining region(HCDR)1, an HCDR2 and an HCDR3, wherein the LCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 30, the LCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 32, the LCDR3 comprises the aminoacid sequence set forth as SEQ ID NO: 34, the HCDR1 comprises the aminoacid sequence set forth as SEQ ID NO: 121, the HCDR2 comprises the aminoacid sequence set forth as SEQ ID NO: 123, and the HCDR3 comprises theamino acid sequence set forth as SEQ ID NO: 248; wherein the sequence ofeach complementarity determining region may differ from the givensequence at up to two amino acid positions, and wherein the monoclonalantibody specifically binds to a ROR1 polypeptide; or the isolatedmonoclonal antibody may comprise a light chain variable domain and aheavy chain variable domain, wherein the light chain variable domaincomprises a light chain complementarity determining region (LCDR)1, anLCDR2 and an LCDR3 and the heavy chain variable domain comprises a heavychain complementarity determining region (HCDR)1, an HCDR2 and an HCDR3,wherein the LCDR1 comprises the amino acid sequence set forth as SEQ IDNO: 24, the LCDR2 comprises the amino acid sequence set forth as SEQ IDNO: 26, the LCDR3 comprises the amino acid sequence set forth as SEQ IDNO: 28, the HCDR1 comprises the amino acid sequence set forth as SEQ IDNO: 115, the HCDR2 comprises the amino acid sequence set forth as SEQ IDNO: 117, and the HCDR3 comprises the amino acid sequence set forth asSEQ ID NO: 252; wherein the sequence of each complementarity determiningregion may differ from the given sequence at up to two amino acidpositions, and wherein the monoclonal antibody specifically binds to aROR1 polypeptide.

As indicated in various embodiments above, the sequence of each CDR maydiffer from the given sequence at up to two amino acid positions. Thismeans that the CDR may contain one or two amino acid substitutionscompared to the given sequence. However, if one or more of the CDRs doescontain amino acid substitutions, the antibody can still selectivelybind to ROR1. Preferably, the amino acid substitutions are conservativesubstitutions.

Preferably, the sequence of each CDR may differ from the given sequenceat one amino acid position. This means that the CDR may contain oneamino acid substitution compared to the given sequence.

Preferably, the amino acid substitution is a conservative substitution.In some embodiment, the CDRs do not contain any amino acidsubstitutions.

In some embodiments above, heavy chain complementarity determiningregion 3 (HCDR3) comprises an amino acid sequence having the sequenceset forth as SEQ ID NO: 248. In such embodiments, HCDR3 preferablycomprises an amino acid sequence as set forth in SEQ ID NO: 125, 246 or247.

In some embodiments above, heavy chain complementarity determiningregion 3 (HCDR3) comprises an amino acid sequence having the sequenceset forth as SEQ ID NO: 252. In such embodiments, HCDR3 preferablycomprises an amino acid sequence as set forth in SEQ ID NO: 119, 249,250 or 251.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (a) may have a light chainvariable domain which comprises a Light Chain Framework Region (LCFR)1comprising the amino acid sequence as set forth as one of SEQ ID NO: 29,SEQ ID NO: 197, SEQ ID NO: 201 or SEQ ID NO: 208; an LCFR2 comprisingthe amino acid sequence as set forth as one of SEQ ID NO: 31, SEQ ID NO:198, SEQ ID NO: 202, SEQ ID NO: 204 or SEQ ID NO: 206; an LCFR3comprising the amino acid sequence as set forth as one of SEQ ID NO: 33,SEQ ID NO: 199, SEQ ID NO: 203 or SEQ ID NO: 207; and an LCFR4comprising the amino acid sequence as set forth as one of SEQ ID NO: 35,SEQ ID NO: 200, SEQ ID NO: 205 or SEQ ID NO: 209.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (a) may have a heavy chainvariable domain which comprises a Heavy Chain Framework Region (HCFR)1comprising the amino acid sequence as set forth as one of SEQ ID NO:120, SEQ ID NO: 181, SEQ ID NO: 188 or SEQ ID NO: 190; an HCFR2comprising the amino acid sequence as set forth as one of SEQ ID NO:122, SEQ ID NO: 182 or SEQ ID NO: 184; an HCFR3 comprising the aminoacid sequence as set forth as one of SEQ ID NO: 124, SEQ ID NO: 183, SEQID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189 or SEQ ID NO: 191; and anHCFR4 comprising the amino acid sequence as set forth as one of SEQ IDNO: 94, SEQ ID NO: 108 or SEQ ID NO: 186.

Preferably the light chain variable domain comprises the amino acidsequence as set forth as one of SEQ ID NO: 80, SEQ ID NO: 210, SEQ IDNO: 211, SEQ ID NO: 212, SEQ ID NO; 213 or SEQ ID NO: 214.

Preferably the heavy chain variable domain comprises the amino acidsequence as set forth as one of SEQ ID NO: 173, SEQ ID NO: 192, SEQ IDNO: 193, SEQ ID NO: 194, SEQ ID NO: 195 or SEQ ID NO: 196.

More preferably,

-   -   (a) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 80 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 173;    -   (b) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 210 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 192;    -   (c) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 211 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 193;    -   (d) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 212 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 194;    -   (e) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 213 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 195; or    -   (f) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 214 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 196.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (b) may have a light chainvariable domain which comprises a Light Chain Framework Region (LCFR)1comprising the amino acid sequence as set forth as one of SEQ ID NO: 23,SEQ ID NO: 230, SEQ ID NO: 238 or SEQ ID NO: 239; an LCFR2 comprisingthe amino acid sequence as set forth as one of SEQ ID NO: 25, SEQ ID NO:231, SEQ ID NO: 233 or SEQ ID NO: 236; an LCFR3 comprising the aminoacid sequence as set forth as one of SEQ ID NO: 27, SEQ ID NO: 232, SEQID NO: 234, SEQ ID NO: 237 or SEQ ID NO: 240; and an LCFR4 comprisingthe amino acid sequence as set forth as one of SEQ ID NO: 14, SEQ ID NO:205 or SEQ ID NO: 234.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (b) may have a heavy chainvariable domain which comprises a Heavy Chain Framework Region (HCFR)1comprising the amino acid sequence as set forth as one of SEQ ID NO:114, SEQ ID NO: 215, SEQ ID NO: 218 or SEQ ID NO: 223; an HCFR2comprising the amino acid sequence as set forth as one of SEQ ID NO:116, SEQ ID NO: 216, SEQ ID NO: 219 or SEQ ID NO: 221; an HCFR3comprising the amino acid sequence as set forth as one of SEQ ID NO:118, SEQ ID NO: 217, SEQ ID NO: 220, SEQ ID NO: 222 or SEQ ID NO: 224;and an HCFR4 comprising the amino acid sequence as set forth as one ofSEQ ID NO: 94, SEQ ID NO: 186 or SEQ ID NO: 108.

Preferably, the light chain variable domain comprises the amino acidsequence as set forth as one of SEQ ID NO: 79, SEQ ID NO: 241, SEQ IDNO: 242, SEQ ID NO: 243, SEQ ID NO: 244 or SEQ ID NO: 245.

Preferably, the heavy chain variable domain comprises the amino acidsequence as set forth as one of SEQ ID NO: 172, SEQ ID NO: 225, SEQ IDNO: 226, SEQ ID NO: 227, SEQ ID NO: 228 or SEQ ID NO: 229.

More preferably,

-   -   (a) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 79 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 172;    -   (b) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 241 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 225;    -   (c) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 242 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 226;    -   (d) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 243 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 227;    -   (e) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 244 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 228; or    -   (f) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 245 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 229.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (c) may have a light chainvariable domain which comprises a Light Chain Framework Region (LCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 8; anLCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 10;an LCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:12; and an LCFR4 comprising the amino acid sequence as set forth as SEQID NO: 14.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (c) may have a heavy chainvariable domain which comprises a Heavy Chain Framework Region (HCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 95; anHCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 97;an HCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:99; and an HCFR4 comprising the amino acid sequence as set forth as SEQID NO: 101.

Preferably, the light chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 76.

Preferably, the heavy chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 169.

More preferably, the light chain variable domain comprises the aminoacid sequence as set forth as SEQ ID NO: 76 and the heavy chain variabledomain comprises the amino acid sequence as set forth as SEQ ID NO: 169.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (d) may have a light chainvariable domain which comprises a Light Chain Framework Region (LCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 36; anLCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 38;an LCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:40; and an LCFR4 comprising the amino acid sequence as set forth as SEQID NO: 42.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (d) may have a heavy chainvariable domain comprises a Heavy Chain Framework Region (HCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 126; anHCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 128;an HCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:130; and an HCFR4 comprising the amino acid sequence as set forth as SEQID NO: 132.

Preferably, the light chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 81.

Preferably, the heavy chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 174.

More preferably, the light chain variable domain comprises the aminoacid sequence as set forth as SEQ ID NO: 81 and the heavy chain variabledomain comprises the amino acid sequence as set forth as SEQ ID NO: 174.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (e) may have a light chainvariable domain which comprises a Light Chain Framework Region (LCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 43; anLCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 45;an LCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:47; and an LCFR4 comprising the amino acid sequence as set forth as SEQID NO: 21.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (e) may have heavy chainvariable domain comprises a Heavy Chain Framework Region (HCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 133; anHCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 135;an HCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:137; and an HCFR4 comprising the amino acid sequence as set forth as SEQID NO: 108.

Preferably, the light chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 82.

Preferably, wherein the heavy chain variable domain comprises the aminoacid sequence as set forth as SEQ ID NO: 175.

More preferably, the light chain variable domain comprises the aminoacid sequence as set forth as SEQ ID NO: 82 and the heavy chain variabledomain comprises the amino acid sequence as set forth as SEQ ID NO: 175.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (f) may have light chainvariable domain which comprises a Light Chain Framework Region (LCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 49; anLCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 51;an LCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:53; and an LCFR4 comprising the amino acid sequence as set forth as SEQID NO: 21.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (f) may have heavy chainvariable domain comprises a Heavy Chain Framework Region (HCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 139; anHCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 141;an HCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:143; and an HCFR4 comprising the amino acid sequence as set forth as SEQID NO: 108.

Preferably, the light chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 83.

Preferably, the heavy chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 176.

More preferably, the light chain variable domain comprises the aminoacid sequence as set forth as SEQ ID NO: 83 and the heavy chain variabledomain comprises the amino acid sequence as set forth as SEQ ID NO: 176.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (g) may have a light chainvariable domain which comprises a Light Chain Framework Region (LCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 55; anLCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 57;an LCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:59; and an LCFR4 comprising the amino acid sequence as set forth as SEQID NO: 61.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (g) may have heavy chainvariable domain which comprises a Heavy Chain Framework Region (HCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 145; anHCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 147;an HCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:149; and an HCFR4 comprising the amino acid sequence as set forth as SEQID NO: 108.

Preferably, wherein the light chain variable domain comprises the aminoacid sequence as set forth as SEQ ID NO: 84.

Preferably, the heavy chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 177.

More preferably, the light chain variable domain comprises the aminoacid sequence as set forth as SEQ ID NO: 84 and the heavy chain variabledomain comprises the amino acid sequence as set forth as SEQ ID NO: 177.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (h) may have light chainvariable domain which comprises a Light Chain Framework Region (LCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 62; anLCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 63;an LCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:64; and an LCFR4 comprising the amino acid sequence as set forth as SEQID NO: 66.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (h) may have a heavy chainvariable domain which comprises a Heavy Chain Framework Region (HCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 151; anHCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 153;an HCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:155; and an HCFR4 comprising the amino acid sequence as set forth as SEQID NO: 108.

Preferably, the light chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 85.

Preferably, the heavy chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 178.

More preferably, the light chain variable domain comprises the aminoacid sequence as set forth as SEQ ID NO: 85 and the heavy chain variabledomain comprises the amino acid sequence as set forth as SEQ ID NO: 178.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (i) may have a light chainvariable domain which comprises a Light Chain Framework Region (LCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 55; anLCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 57;an LCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:59; and an LCFR4 comprising the amino acid sequence as set forth as SEQID NO: 69.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (i) may have a heavy chainvariable domain which comprises a Heavy Chain Framework Region (HCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 157; anHCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 159;an HCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:161; and an HCFR4 comprising the amino acid sequence as set forth as SEQID NO: 108.

Preferably, the light chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 86.

Preferably, the heavy chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 179.

More preferably, the light chain variable domain comprises the aminoacid sequence as set forth as SEQ ID NO: 86 and the heavy chain variabledomain comprises the amino acid sequence as set forth as SEQ ID NO: 179.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (j) may have a light chainvariable domain which comprises a Light Chain Framework Region (LCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 70; anLCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 72;an LCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:73; and an LCFR4 comprising the amino acid sequence as set forth as SEQID NO: 21.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (j) may have heavy chainvariable domain which comprises a Heavy Chain Framework Region (HCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 114; anHCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 164;an HCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:166; and an HCFR4 comprising the amino acid sequence as set forth as SEQID NO: 108.

Preferably, the light chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 87.

Preferably, the heavy chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 180.

More preferably, the light chain variable domain comprises the aminoacid sequence as set forth as SEQ ID NO: 87 and the heavy chain variabledomain comprises the amino acid sequence as set forth as SEQ ID NO: 180.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (k) may have a light chainvariable domain which comprises a Light Chain Framework Region (LCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 1; anLCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 3;an LCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:5; and an LCFR4 comprising the amino acid sequence as set forth as SEQID NO: 7.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (k) may have heavy chainvariable domain which comprises a Heavy Chain Framework Region (HCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 88; anHCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 90;an HCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:92; and an HCFR4 comprising the amino acid sequence as set forth as SEQID NO: 94.

Preferably, the light chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 75.

Preferably, the heavy chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 168.

More preferably, the light chain variable domain comprises the aminoacid sequence as set forth as SEQ ID NO: 75 and the heavy chain variabledomain comprises the amino acid sequence as set forth as SEQ ID NO: 168.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (1) may have a light chainvariable domain which comprises a Light Chain Framework Region (LCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 15; anLCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 17;an LCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:19; and an LCFR4 comprising the amino acid sequence as set forth as SEQID NO: 21.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (1) may have heavy chainvariable domain which comprises a Heavy Chain Framework Region (HCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 102; anHCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 104;an HCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:106; and an HCFR4 comprising the amino acid sequence as set forth as SEQID NO: 108.

Preferably, the light chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 77.

Preferably, the heavy chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 170.

More preferably, the light chain variable domain comprises the aminoacid sequence as set forth as SEQ ID NO: 77 and the heavy chain variabledomain comprises the amino acid sequence as set forth as SEQ ID NO: 170.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (m) may have a light chainvariable domain which comprises a Light Chain Framework Region (LCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 15; anLCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 17;an LCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:19; and an LCFR4 comprising the amino acid sequence as set forth as SEQID NO: 21.

The isolated monoclonal antibody according to the preferred andparticularly preferred embodiments part (m) may have heavy chainvariable domain which comprises a Heavy Chain Framework Region (HCFR)1comprising the amino acid sequence as set forth as SEQ ID NO: 109; anHCFR2 comprising the amino acid sequence as set forth as SEQ ID NO: 111;an HCFR3 comprising the amino acid sequence as set forth as SEQ ID NO:112; and an HCFR4 comprising the amino acid sequence as set forth as SEQID NO: 108.

Preferably, the light chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 78.

Preferably, the heavy chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 171.

More preferably, the light chain variable domain comprises the aminoacid sequence as set forth as SEQ ID NO: 78 and the heavy chain variabledomain comprises the amino acid sequence as set forth as SEQ ID NO: 171.

In particular embodiments, the light chain variable domain comprises theamino acid sequence as set forth as one of SEQ ID NOs: 80, 210, 211,212, 213 and 214. More preferably, the light chain variable domaincomprises the amino acid sequence as set forth as one of SEQ ID NOs:210, 211, 212, 213 and 214.

Preferably, the heavy chain variable domain comprises the amino acidsequence as set forth as one of SEQ ID NOs: 173, 192, 193, 194, 195 and196. More preferably, the heavy chain variable domain comprises theamino acid sequence as set forth as one of SEQ ID NOs: 192, 193, 194,195 and 196.

SEQ ID NOs: 210, 211, 212, 213 and 214 are humanised light chainvariable regions produced from clone A. SEQ ID NOs: 192, 193, 194, 195and 196 are humanised heavy chain variable regions produced from cloneA. The combination of these light and heavy chain regions results in 25different constructs.

Therefore, in some embodiments, the light chain variable domaincomprises the amino acid sequence as set forth as SEQ ID NO: 210 and theheavy chain variable domain comprises the amino acid sequence as setforth as one of SEQ ID NOs: 192, 193, 194, 195 and 196.

In other embodiments, the light chain variable domain comprises theamino acid sequence as set forth as SEQ ID NO: 211 and the heavy chainvariable domain comprises the amino acid sequence as set forth as one ofSEQ ID NOs: 192, 193, 194, 195 and 196.

In further embodiments, the light chain variable domain comprises theamino acid sequence as set forth as SEQ ID NO: 212 and the heavy chainvariable domain comprises the amino acid sequence as set forth as one ofSEQ ID NOs: 192, 193, 194, 195 and 196.

In alternative embodiments, the light chain variable domain comprisesthe amino acid sequence as set forth as SEQ ID NO: 213 and the heavychain variable domain comprises the amino acid sequence as set forth asone of SEQ ID NOs: 192, 193, 194, 195 and 196.

In various embodiments, the light chain variable domain comprises theamino acid sequence as set forth as SEQ ID NO: 214 and the heavy chainvariable domain comprises the amino acid sequence as set forth as one ofSEQ ID NOs: 192, 193, 194, 195 and 196.

Similarly, in some embodiments, the heavy chain variable domaincomprises the amino acid sequence as set forth as SEQ ID NO: 192 and thelight chain variable domain comprises the amino acid sequence as setforth as one of SEQ ID NOs: 210, 211, 212, 213 and 214.

In other embodiments, the heavy chain variable domain comprises theamino acid sequence as set forth as SEQ ID NO: 193 and the light chainvariable domain comprises the amino acid sequence as set forth as one ofSEQ ID NOs: 210, 211, 212, 213 and 214.

In further embodiments, the heavy chain variable domain comprises theamino acid sequence as set forth as SEQ ID NO: 194 and the light chainvariable domain comprises the amino acid sequence as set forth as one ofSEQ ID NOs: 210, 211, 212, 213 and 214.

In alternative embodiments, the heavy chain variable domain comprisesthe amino acid sequence as set forth as SEQ ID NO: 195 and the lightchain variable domain comprises the amino acid sequence as set forth asone of SEQ ID NOs: 210, 211, 212, 213 and 214.

In various embodiments, the heavy chain variable domain comprises theamino acid sequence as set forth as SEQ ID NO: 196 and the light chainvariable domain comprises the amino acid sequence as set forth as one ofSEQ ID NOs: 210, 211, 212, 213 and 214.

In certain embodiments, the light chain variable domain comprises theamino acid sequence as set forth as one of SEQ ID NOs: 79, 241, 242,243, 244 and 245. More preferably, the light chain variable domaincomprises the amino acid sequence as set forth as one of SEQ ID NOs:241, 242, 243, 244 and 245.

Preferably, the heavy chain variable domain comprises the amino acidsequence as set forth as one of SEQ ID NOs: 172, 225, 226, 227, 228 and229. More preferably, the heavy chain variable domain comprises theamino acid sequence as set forth as one of SEQ ID NOs: 225, 226, 227,228 and 229.

SEQ ID NOs: 241, 242, 243, 244 and 245 are humanised light chainvariable regions produced from clone F. SEQ ID NOs: 225, 226, 227, 228and 229 are humanised heavy chain variable regions produced from cloneF. The combination of these light and heavy chain regions results in 25different constructs.

Therefore, in some embodiments, the light chain variable domaincomprises the amino acid sequence as set forth as SEQ ID NO: 241 and theheavy chain variable domain comprises the amino acid sequence as setforth as one of SEQ ID NOs: 225, 226, 227, 228 and 229.

In other embodiments, the light chain variable domain comprises theamino acid sequence as set forth as SEQ ID NO: 242 and the heavy chainvariable domain comprises the amino acid sequence as set forth as one ofSEQ ID NOs: 225, 226, 227, 228 and 229.

In further embodiments, the light chain variable domain comprises theamino acid sequence as set forth as SEQ ID NO: 243 and the heavy chainvariable domain comprises the amino acid sequence as set forth as one ofSEQ ID NOs: 225, 226, 227, 228 and 229.

In alternative embodiments, the light chain variable domain comprisesthe amino acid sequence as set forth as SEQ ID NO: 244 and the heavychain variable domain comprises the amino acid sequence as set forth asone of SEQ ID NOs: 225, 226, 227, 228 and 229.

In various embodiments, the light chain variable domain comprises theamino acid sequence as set forth as SEQ ID NO: 245 and the heavy chainvariable domain comprises the amino acid sequence as set forth as one ofSEQ ID NOs: 225, 226, 227, 228 and 229.

Similarly, in some embodiments, the heavy chain variable domaincomprises the amino acid sequence as set forth as SEQ ID NO: 225 and thelight chain variable domain comprises the amino acid sequence as setforth as one of SEQ ID NOs: 241, 242, 243, 244 and 245.

In other embodiments, the heavy chain variable domain comprises theamino acid sequence as set forth as SEQ ID NO: 226 and the light chainvariable domain comprises the amino acid sequence as set forth as one ofSEQ ID NOs: 241, 242, 243, 244 and 245.

In further embodiments, the heavy chain variable domain comprises theamino acid sequence as set forth as SEQ ID NO: 227 and the light chainvariable domain comprises the amino acid sequence as set forth as one ofSEQ ID NOs: 241, 242, 243, 244 and 245.

In alternative embodiments, the heavy chain variable domain comprisesthe amino acid sequence as set forth as SEQ ID NO: 228 and the lightchain variable domain comprises the amino acid sequence as set forth asone of SEQ ID NOs: 241, 242, 243, 244 and 245.

In various embodiments, the heavy chain variable domain comprises theamino acid sequence as set forth as SEQ ID NO: 229 and the light chainvariable domain comprises the amino acid sequence as set forth as one ofSEQ ID NOs: 241, 242, 243, 244 and 245.

As indicated below, the sequence of each light chain variable domain andheavy chain variable domain referred to above may differ from the givensequence. For example, the light/heavy chain variable domain maycomprise a sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the amino acid sequences as set forth inthe sequence listing.

Alternatively, the light/heavy chain variable domain sequence may differat up to 10 amino acid positions, although it is preferred that fewerthan 10 amino acid substitutions are present so that there may be up to9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.

In the embodiments referred to above, the Light Chain Framework Regions,the Heavy Chain Framework Regions, the Light Chain Variable Domains andthe Heavy Chain Variable Domains may comprise an amino acid sequence atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical tothe amino acid sequences as set forth above. For example, the LightChain Framework Regions, the Heavy Chain Framework Regions, the LightChain Variable Domains and the Heavy Chain Variable Domains may includeat most 10, at most 9, at most 8, at most 7, at most 6, at most 5, atmost 4, at most 3, at most two or at most one amino acid substitutionsin the amino acid sequences as set forth above. Where there is variationin the sequences of the Light Chain Variable Domain and the Heavy ChainVariable Domain, any amino acid substitutions are preferably not in theCDRs. In particular, the Light Chain Framework Regions and/or the HeavyChain Framework Regions of the antibodies described above may comprisean amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the amino acid sequences as set forth above.Further, the Light Chain Framework Regions and/or the Heavy ChainFramework Regions may include at most 10, at most 9, at most 8, at most7, at most 6, at most 5, at most 4, at most 3, at most two or at mostone amino acid substitutions in the amino acid sequences as set forthabove. Preferably the amino acid substitutions are conservativesubstitutions as described above. For example, the framework regions maycomprise such substitutions in order to humanise the sequence.Preferably, the framework regions are humanised.

In a second embodiment, the isolated monoclonal antibody comprises alight chain variable domain and a heavy chain variable domain, whereinthe light chain variable domain comprises a light chain complementaritydetermining region (LCDR)1, an LCDR2 and an LCDR3 and the heavy chainvariable domain comprises a heavy chain complementarity determiningregion (HCDR)1, an HCDR2 and an HCDR3, wherein

-   -   (a) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 277, the LCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 278, the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 34, the HCDR1 comprises the        amino acid sequence set forth as SEQ ID NO: 279, the HCDR2        comprises the amino acid sequence set forth as SEQ ID NO: 280,        and the HCDR3 comprises the amino acid sequence set forth as SEQ        ID NO: 281;    -   (b) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 272, the LCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 273, the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 28, the HCDR1 comprises the        amino acid sequence set forth as SEQ ID NO: 274, the HCDR2        comprises the amino acid sequence set forth as SEQ ID NO: 275,        and the HCDR3 comprises the amino acid sequence set forth as SEQ        ID NO: 276;    -   (c) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 260, the LCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 261, the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 13, the HCDR1 comprises the        amino acid sequence set forth as SEQ ID NO: 262, the HCDR2        comprises the amino acid sequence set forth as SEQ ID NO: 263,        and the HCDR3 comprises the amino acid sequence set forth as SEQ        ID NO: 264;    -   (d) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 282, the LCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 283, the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 41, the HCDR1 comprises the        amino acid sequence set forth as SEQ ID NO: 284, the HCDR2        comprises the amino acid sequence set forth as SEQ ID NO: 285,        and the HCDR3 comprises the amino acid sequence set forth as SEQ        ID NO: 286;    -   (e) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 287, the LCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 288, the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 48, the HCDR1 comprises the        amino acid sequence set forth as SEQ ID NO: 289, the HCDR2        comprises the amino acid sequence set forth as SEQ ID NO: 290,        and the HCDR3 comprises the amino acid sequence set forth as SEQ        ID NO: 291;    -   (f) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 292, the LCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 293, the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 54, the HCDR1 comprises the        amino acid sequence set forth as SEQ ID NO: 294, the HCDR2        comprises the amino acid sequence set forth as SEQ ID NO: 295,        and the HCDR3 comprises the amino acid sequence set forth as SEQ        ID NO: 296;    -   (g) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 297, the LCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 298, the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 60, the HCDR1 comprises the        amino acid sequence set forth as SEQ ID NO: 299, the HCDR2        comprises the amino acid sequence set forth as SEQ ID NO: 300,        and the HCDR3 comprises the amino acid sequence set forth as SEQ        ID NO: 301;    -   (h) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 287, the LCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 302, the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 65, the HCDR1 comprises the        amino acid sequence set forth as SEQ ID NO: 289, the HCDR2        comprises the amino acid sequence set forth as SEQ ID NO: 303,        and the HCDR3 comprises the amino acid sequence set forth as SEQ        ID NO: 304;    -   (i) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 305, the LCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 298, the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 68, the HCDR1 comprises the        amino acid sequence set forth as SEQ ID NO: 306, the HCDR2        comprises the amino acid sequence set forth as SEQ ID NO: 307,        and the HCDR3 comprises the amino acid sequence set forth as SEQ        ID NO: 308;    -   (j) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 309, the LCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 310, the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 74, the HCDR1 comprises the        amino acid sequence set forth as SEQ ID NO: 311, the HCDR2        comprises the amino acid sequence set forth as SEQ ID NO: 312,        and the HCDR3 comprises the amino acid sequence set forth as SEQ        ID NO: 313;    -   (k) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 255, the LCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 256, the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 6, the HCDR1 comprises the        amino acid sequence set forth as SEQ ID NO: 257, the HCDR2        comprises the amino acid sequence set forth as SEQ ID NO: 258,        and the HCDR3 comprises the amino acid sequence set forth as SEQ        ID NO: 259;    -   (1) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 265, the LCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 266, the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 20, the HCDR1 comprises the        amino acid sequence set forth as SEQ ID NO: 267, the HCDR2        comprises the amino acid sequence set forth as SEQ ID NO: 268,        and the HCDR3 comprises the amino acid sequence set forth as SEQ        ID NO: 269; or    -   (m) the LCDR1 comprises the amino acid sequence set forth as SEQ        ID NO: 270, the LCDR2 comprises the amino acid sequence set        forth as SEQ ID NO: 266, the LCDR3 comprises the amino acid        sequence set forth as SEQ ID NO: 20, the HCDR1 comprises the        amino acid sequence set forth as SEQ ID NO: 267, the HCDR2        comprises the amino acid sequence set forth as SEQ ID NO: 268,        and the HCDR3 comprises the amino acid sequence set forth as SEQ        ID NO: 271;    -   wherein the sequence of each complementarity determining region        may differ from the given sequence at up to two amino acid        positions, and    -   wherein the monoclonal antibody specifically binds to a ROR1        polypeptide.

As indicated in the embodiment above, the sequence of each CDR maydiffer from the given sequence at up to two amino acid positions. Thismeans that the CDR may contain one or two amino acid substitutionscompared to the given sequence. However, if one or more of the CDRs doescontain amino acid substitutions, the antibody can still selectivelybind to ROR1. Preferably, the amino acid substitutions are conservativesubstitutions.

Preferably, the sequence of each CDR may differ from the given sequenceat one amino acid position. This means that the CDR may contain oneamino acid substitution compared to the given sequence. Preferably, theamino acid substitution is a conservative substitution. In someembodiments, the CDRs do not contain any amino acid substitutions.

The isolated monoclonal antibody according to part (a) of the secondembodiment may have a light chain variable domain which comprises theamino acid sequence as set forth as one of SEQ ID NO: 80, SEQ ID NO:210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO; 213 or SEQ ID NO: 214.The heavy chain variable domain may comprise the amino acid sequence asset forth as one of SEQ ID NO: 173, SEQ ID NO: 192, SEQ ID NO: 193, SEQID NO: 194, SEQ ID NO: 195 or SEQ ID NO: 196.

More preferably,

-   -   (a) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 80 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 173;    -   (b) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 210 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 192;    -   (c) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 211 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 193;    -   (d) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 212 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 194;    -   (e) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 213 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 195; or    -   (f) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 214 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 196.

The isolated monoclonal antibody according to the part (b) of the secondembodiment may have a light chain variable domain which comprises theamino acid sequence as set forth as one of SEQ ID NO: 79, SEQ ID NO:241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244 or SEQ ID NO: 245.The heavy chain variable domain may comprise the amino acid sequence asset forth as one of SEQ ID NO: 172, SEQ ID NO: 225, SEQ ID NO: 226, SEQID NO: 227, SEQ ID NO: 228 or SEQ ID NO: 229.

More preferably,

-   -   (a) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 79 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 172;    -   (b) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 241 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 225;    -   (c) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 242 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 226;    -   (d) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 243 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 227;    -   (e) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 244 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 228; or    -   (f) the light chain variable domain comprises the amino acid        sequence as set forth as SEQ ID NO: 245 and the heavy chain        variable domain comprises the amino acid sequence as set forth        as SEQ ID NO: 229.

The isolated monoclonal antibody according to the part (c) of the secondembodiment may have a light chain variable domain which comprises theamino acid sequence as set forth as SEQ ID NO: 76. The heavy chainvariable domain may comprise the amino acid sequence as set forth as SEQID NO: 169.

The isolated monoclonal antibody according to the part (d) of the secondembodiment may have a light chain variable domain which comprises theamino acid sequence as set forth as SEQ ID NO: 81. The heavy chainvariable domain comprises the amino acid sequence as set forth as SEQ IDNO: 174.

The isolated monoclonal antibody according to the part (e) of the secondembodiment may have a light chain variable domain which comprises theamino acid sequence as set forth as SEQ ID NO: 82. The heavy chainvariable domain may comprise the amino acid sequence as set forth as SEQID NO: 175.

The isolated monoclonal antibody according to the part (f) of the secondembodiment may have a light chain variable domain which comprises theamino acid sequence as set forth as SEQ ID NO: 83. The heavy chainvariable domain may comprise the amino acid sequence as set forth as SEQID NO: 176.

The isolated monoclonal antibody according to the part (g) of the secondembodiment may have a light chain variable domain which comprises theamino acid sequence as set forth as SEQ ID NO: 84. The heavy chainvariable domain may comprise the amino acid sequence as set forth as SEQID NO: 177.

The isolated monoclonal antibody according to the part (h) of the secondembodiment may have a light chain variable domain which comprises theamino acid sequence as set forth as SEQ ID NO: 85. The heavy chainvariable domain may comprise the amino acid sequence as set forth as SEQID NO: 178.

The isolated monoclonal antibody according to the part (i) of the secondembodiment may have a light chain variable domain which comprises theamino acid sequence as set forth as SEQ ID NO: 86. The heavy chainvariable domain may comprise the amino acid sequence as set forth as SEQID NO: 179.

The isolated monoclonal antibody according to the part (j) of the secondembodiment may have a light chain variable domain which comprises theamino acid sequence as set forth as SEQ ID NO: 87. The heavy chainvariable domain may comprise the amino acid sequence as set forth as SEQID NO: 180.

The isolated monoclonal antibody according to the part (k) of the secondembodiment may have a light chain variable domain which comprises theamino acid sequence as set forth as SEQ ID NO: 75. The heavy chainvariable domain may comprise the amino acid sequence as set forth as SEQID NO: 168.

The isolated monoclonal antibody according to the part (1) of the secondembodiment may have a light chain variable domain which comprises theamino acid sequence as set forth as SEQ ID NO: 77. The heavy chainvariable domain may comprise the amino acid sequence as set forth as SEQID NO: 170.

The isolated monoclonal antibody according to the part (m) of the secondembodiment may have a light chain variable domain which comprises theamino acid sequence as set forth as SEQ ID NO: 78. The heavy chainvariable domain may comprise the amino acid sequence as set forth as SEQID NO: 171.

The sequence of each light chain variable domain and heavy chainvariable domain referred to above for the second embodiment may differfrom the given sequence. For example, the light/heavy chain variabledomain may comprise a sequence which is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences asset forth in the sequence listing. Alternatively, the light/heavy chainvariable domain sequence may differ at up to 10 amino acid positions,although it is preferred that fewer than 10 amino acid substitutions arepresent so that there may be up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 aminoacid substitutions.

With reference to all the embodiments described above, one skilled inthe art will be aware that any substitutions will retain critical aminoacid residues necessary for correct folding and stabilizing between theV_(H) and the V_(L) regions, and will retain the charge characteristicsof the residues in order to preserve the low pI and low toxicity of themolecules. Thus, one of skill in the art can readily review thesequences shown above, identify a conservative substitution, and producethe conservative variant using well-known molecular techniques.

Epitope mapping has been carried out for some of the antibodiesdiscussed above. In one embodiment, it has been found that residuesAsn-47 and Ile-48 of human ROR1 are essential for antibody binding.Therefore, there is provided a monoclonal antibody that binds to anepitope of ROR1, wherein the epitope comprises amino acids Asn-47 and/orIle-48. Preferably, the epitope comprises amino acids Asn-47 and Ile-48.In some embodiments, the epitope may comprise amino acids Asn-47,Ile-48, Ser-49, Ser-50 and Glu-51 (NISSE—SEQ ID NO: 253).

In another embodiment, it has been found that residue Gln-261 of humanROR1 is essential for antibody binding. Therefore, there is alsoprovided a monoclonal antibody that binds to an epitope of ROR1, whereinthe epitope comprises amino acid Gln-261.

Antibody fragments are encompassed by the present disclosure, such asFab, F(ab′)₂, and Fv which include a heavy chain and light chainvariable region and are capable of binding the epitopic determinant onROR1. These antibody fragments retain the ability to selectively bindwith the antigen and are described above. The fragments can be includedin a bispecific antibody. Methods of making these fragments are known inthe art (see for example, Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, New York, 1988).

In a further group of embodiments, the antibodies are Fv antibodies,which are typically about 25 kDa and contain a complete antigen-bindingsite with three CDRs per each heavy chain and each light chain. Toproduce these antibodies, the V_(H) and the V_(L) can be expressed fromtwo individual nucleic acid constructs in a host cell. If the V_(H) andthe V_(L) are expressed non-contiguously, the chains of the Fv antibodyare typically held together by noncovalent interactions. However, thesechains tend to dissociate upon dilution, so methods have been developedto crosslink the chains through glutaraldehyde, intermoleculardisulfides, or a peptide linker. Thus, in one example, the Fv can be adisulfide stabilized Fv (dsFv), wherein the heavy chain variable regionand the light chain variable region are chemically linked by disulfidebonds.

In an additional example, the Fv fragments comprise V_(H) and V_(L)chains connected by a peptide linker. These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains connectedby an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are known in the art (see Whitlow et al.,Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991;Bird et al., Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack etal., Bio/Technology 11:1271, 1993; and Sandhu, supra). Dimers of asingle chain antibody (scFV₂), are also contemplated.

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli of DNA encoding the fragment.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly (see U.S. Pat. Nos. 4,036,945 and 4,331,647,and references contained therein; Nisonhoff et al., Arch. Biochem.Biophys. 89:230, 1960; Porter, Biochem. J 73:119, 1959; Edelman et al.,Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; andColigan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

The antibodies or antibody fragments disclosed herein can be derivatizedor linked to another molecule (such as another peptide or protein). Ingeneral, the antibody or portion thereof is derivatized such that thebinding to the ROR1 polypeptide is not affected adversely by thederivatization or labeling. For example, the antibody can befunctionally linked, for example, by chemical coupling, genetic fusion,noncovalent association or otherwise to one or more other molecularentities, such as another antibody (for example, a bispecific antibodyor a diabody), a detection agent, a pharmaceutical agent, and/or aprotein or peptide that can mediate associate of the antibody orantibody portion with another molecule (such as a streptavidin coreregion or a polyhistidine tag).

One type of derivatized antibody is produced by cross-linking two ormore antibodies (of the same type or of different types, such as tocreate bispecific antibodies). Suitable crosslinkers include those thatare heterobifunctional, having two distinctly reactive groups separatedby an appropriate spacer (such asm-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (suchas disuccinimidyl suberate). Such linkers are available from PierceChemical Company (Rockford, Ill.).

An antibody that specifically binds a ROR1 polypeptide can be labeledwith a detectable moiety or marker as described above.

An antibody can also be labeled with a radiolabeled amino acid. Examplesof radiolabels include, but are not limited to, the followingradioisotopes or radionucleotides: ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I. The radiolabel may be used for both diagnostic andtherapeutic purposes.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

An antibody can also be derivatized with a chemical group such aspolyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrategroup. These groups may be useful to improve the biologicalcharacteristics of the antibody, such as to increase serum half-life orto increase tissue binding.

Polynucleotides and Expression

Nucleotide sequences encoding an antibody that specifically binds a ROR1polypeptide are also provided. The antibody can specifically bind ROR1.Expression vectors are also provided for their efficient expression incells (for example, mammalian cells).

Recombinant expression of an antibody generally requires construction ofan expression vector containing a polynucleotide that encodes theantibody or antibody fragment. Replicable vectors are provided includinga nucleotide sequence encoding an antibody molecule, a heavy or lightchain of an antibody, a heavy or light chain variable domain of anantibody or a portion thereof, or a heavy or light chain CDR, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of an antibody molecule (see, e.g., U.S.Pat. Nos. 5,981,216; 5,591,639; 5,658,759 and 5,122,464) and thevariable domain of the antibody may be cloned into such a vector forexpression of the entire heavy, the entire light chain, or both theentire heavy and light chains.

Nucleic acid molecules (also referred to as polynucleotides) encodingthe polypeptides provided herein (including, but not limited toantibodies) can readily be produced by one of skill in the art. Forexample, these nucleic acids can be produced using the amino acidsequences provided herein (such as the CDR sequences, heavy chain andlight chain sequences), sequences available in the art (such asframework sequences), and the genetic code.

One of skill in the art can readily use the genetic code to construct avariety of functionally equivalent nucleic acids, such as nucleic acidswhich differ in sequence but which encode the same antibody sequence, orencode a conjugate or fusion protein including the V_(L) and/or V_(H)nucleic acid sequence.

Nucleic acid sequences encoding the antibodies that specifically bind aROR1 poypeptide, can be prepared by any suitable method including, forexample, cloning of appropriate sequences or by direct chemicalsynthesis by methods such as the phosphotriester method of Narang etal., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brownet al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramiditemethod of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solidphase phosphoramidite triester method described by Beaucage & Caruthers,Tetra. Letts. 22(20):1859-1862, 1981, for example, using an automatedsynthesizer as described in, for example, Needham-VanDevanter et al.,Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method ofU.S. Pat. No. 4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is generally limited tosequences of about 100 bases, longer sequences may be obtained by theligation of shorter sequences.

Exemplary nucleic acids can be prepared by cloning techniques. Examplesof appropriate cloning and sequencing techniques, and instructionssufficient to direct persons of skill through many cloning exercises arefound in Sambrook et al., supra, Berger and Kimmel (eds.), supra, andAusubel, supra. Product information from manufacturers of biologicalreagents and experimental equipment also provide useful information.Such manufacturers include the SIGMA Chemical Company (Saint Louis,Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway,N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem GenesCorp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc.,GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), FlukaChemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),Invitrogen (Carlsbad, Calif.), and Applied Biosystems (Foster City,Calif.), as well as many other commercial sources known to one of skill.

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

Any of the nucleic acids encoding any of the antibodies, V_(H) and/orV_(L), disclosed herein (or fragment thereof) can be expressed in arecombinantly engineered cell such as bacteria, plant, yeast, insect andmammalian cells. These antibodies can be expressed as individual V_(H)and/or V_(L) chain, or can be expressed as a fusion protein. Animmunoadhesin can also be expressed. Thus, in some examples, nucleicacids encoding a V_(H) and V_(L), and immunoadhesin are provided. Thenucleic acid sequences can optionally encode a leader sequence.

To create a single chain antibody, (scFv) the V_(H)- and V_(L)-encodingDNA fragments are operatively linked to another fragment encoding aflexible linker, e.g., encoding the amino acid sequence (Gly₄-Ser)₃,such that the V_(H) and V_(L) sequences can be expressed as a contiguoussingle-chain protein, with the V_(L) and V_(H) domains joined by theflexible linker (see, e.g., Bird et al., Science 242:423-426, 1988;Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; McCaffertyet al., Nature 348:552-554, 1990). Optionally, a cleavage site can beincluded in a linker, such as a furin cleavage site.

The nucleic acid encoding the V_(H) and/or the V_(L) optionally canencode an Fc domain (immunoadhesin). The Fc domain can be an IgA, IgM orIgG Fc domain. The Fc domain can be an optimized Fc domain, as describedin U.S. Published Patent Application No. 20100/093979, incorporatedherein by reference. In one example, the immunoadhesin is an IgG₁ Fc.

The single chain antibody may be monovalent, if only a single V_(H) andV_(L) are used, bivalent, if two V_(H) and V_(L) are used, orpolyvalent, if more than two V_(H) and V_(L) are used. Bispecific orpolyvalent antibodies may be generated that bind specifically to a ROR1polypeptide and another antigen. The encoded V_(H) and V_(L) optionallycan include a furin cleavage site between the V_(H) and V_(L) domains.

It is expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of proteinsincluding E. coli, other bacterial hosts, yeast, and various highereukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines. Oncethe expression vector is transferred to a host cell by conventionaltechniques, the transfected cells are then cultured by conventionaltechniques, such as to produce an antibody. Thus, host cells areprovided containing a polynucleotide encoding an antibody or fragmentsthereof, or a heavy or light chain thereof, or portion thereof, or asingle-chain antibody of the invention, operably linked to aheterologous promoter. In certain embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

Mammalian cell lines available as hosts for expression of recombinantantibodies are well known in the art and include many immortalized celllines available from the American Type Culture Collection (ATCC),including but not limited to Chinese hamster ovary (CHO) cells, HeLacells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney293 cells, and a number of other cell lines. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the antibody or portion thereofexpressed. To this end, eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Suchmammalian host cells include but are not limited to CHO, VERY, BHK,Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0(a murine myeloma cell line that does not endogenously produce anyfunctional immunoglobulin chains), SP20, CRL7O3O and HsS78Bst cells. Inone embodiment, human cell lines are of use. In one embodiment, thehuman cell line PER.C6. (Crucell, Netherlands) can be used. Additionalcell lines which may be used as hosts for expression of recombinantantibodies include, but are not limited to, insect cells (e.g. Sf21/Sf9,Trichoplusia ni Bti-Tn5b1-4) or yeast cells (e.g. S. cerevisiae, Pichia,U.S. Pat. No. 7,326,681), plant cells (US Published Patent ApplicationNo. 20080066200); and chicken cells (PCT Publication No. WO2008142124).

The host cell can be a gram positive bacteria including, but not limitedto, Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Methods for expressing protein in gram positivebacteria, such as Lactobaccillus are well known in the art, see forexample, U.S. Published Patent Application No. 20100/080774. Expressionvectors for lactobacillus are described, for example in U.S. Pat. Nos.6,100,388, and 5,728,571. Leader sequences can be included forexpression in Lactobacillus. Gram negative bacteria include, but notlimited to, E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, andUreaplasma.

One or more DNA sequences encoding the antibody or fragment thereof canbe expressed in vitro by DNA transfer into a suitable host cell. Theterm also includes any progeny of the subject host cell. It isunderstood that all progeny may not be identical to the parental cellsince there may be mutations that occur during replication. Methods ofstable transfer, meaning that the foreign DNA is continuously maintainedin the host, are known in the art.

The expression of nucleic acids encoding the isolated proteins describedherein can be achieved by operably linking the DNA to a promoter (whichis either constitutive or inducible), followed by incorporation into anexpression cassette. The promoter can be any promoter of interest,including a cytomegalovirus promoter and a human T cell lymphotrophicvirus promoter (HTLV)-1. Optionally, an enhancer, such as acytomegalovirus enhancer, is included in the construct. The cassettescan be suitable for replication and integration in either prokaryotes oreukaryotes. Typical expression cassettes contain specific sequencesuseful for regulation of the expression of the DNA encoding the protein.For example, the expression cassettes can include appropriate promoters,enhancers, transcription and translation terminators, initiationsequences, a start codon (i.e., ATG) in front of a protein-encodinggene, splicing signal for introns, sequences for the maintenance of thecorrect reading frame of that gene to permit proper translation of mRNA,and stop codons. The vector can encode a selectable marker, such as amarker encoding drug resistance (for example, ampicillin or tetracyclineresistance).

To obtain high level expression of a cloned gene, it is desirable toconstruct expression cassettes which contain, at the minimum, a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation (internal ribosomal binding sequences), and atranscription/translation terminator. For E. coli, this includes apromoter such as the T7, trp, lac, or lambda promoters, a ribosomebinding site, and preferably a transcription termination signal. Foreukaryotic cells, the control sequences can include a promoter and/or anenhancer derived from, for example, an immunoglobulin gene, HTLV, SV40or cytomegalovirus, and a polyadenylation sequence, and can furtherinclude splice donor and/or acceptor sequences (for example, CMV and/orHTLV splice acceptor and donor sequences). The cassettes can betransferred into the chosen host cell by well-known methods such astransformation or electroporation for E. coli and calcium phosphatetreatment, electroporation or lipofection for mammalian cells. Cellstransformed by the cassettes can be selected by resistance toantibiotics conferred by genes contained in the cassettes, such as theamp, gpt, neo and hyg genes.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the antibody,labeled antibody, or functional fragment thereof, and a second foreignDNA molecule encoding a selectable phenotype, such as the herpes simplexthymidine kinase gene. Another method is to use a eukaryotic viralvector, such as simian virus 40 (SV40) or bovine papilloma virus, totransiently infect or transform eukaryotic cells and express the protein(see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982). One of skill in the art can readily usean expression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the COS,CHO, HeLa and myeloma cell lines.

Modifications can be made to a nucleic acid encoding a polypeptidedescribed herein without diminishing its biological activity. Somemodifications can be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, termination codons, a methionine added at the aminoterminus to provide an initiation, site, additional amino acids placedon either terminus to create conveniently located restriction sites, oradditional amino acids (such as poly His) to aid in purification steps.In addition to recombinant methods, the immunoconjugates, effectormoieties, and antibodies of the present disclosure can also beconstructed in whole or in part using standard peptide synthesis wellknown in the art.

Once expressed, the recombinant immunoconjugates, antibodies, and/oreffector molecules can be purified according to standard procedures ofthe art, including ammonium sulfate precipitation, affinity columns,column chromatography, and the like (see, generally, R. Scopes, PROTEINPURIFICATION, Springer-Verlag, N.Y., 1982). The antibodies,immunoconjugates and effector molecules need not be 100% pure. Oncepurified, partially or to homogeneity as desired, if to be usedtherapeutically, the polypeptides should be substantially free ofendotoxin.

Methods for expression of antibodies and/or refolding to an appropriateactive form, including single chain antibodies, from bacteria such as E.coli have been described and are well-known and are applicable to theantibodies disclosed herein. See, Buchner et al., Anal. Biochem.205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991; Huse et al.,Science 246:1275, 1989 and Ward et al., Nature 341:544, 1989.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena et al., Biochemistry 9: 5015-5021, 1970, andespecially as described by Buchner et al., supra.

Renaturation is typically accomplished by dilution (for example,100-fold) of the denatured and reduced protein into refolding buffer. Anexemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. An exemplary yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5-foldmolar excess of one protein over the other is not exceeded. Excessoxidized glutathione or other oxidizing low molecular weight compoundscan be added to the refolding solution after the redox-shuffling iscompleted.

In addition to recombinant methods, the antibodies, labeled antibodiesand functional fragments thereof that are disclosed herein can also beconstructed in whole or in part using standard peptide synthesis. Solidphase synthesis of the polypeptides of less than about 50 amino acids inlength can be accomplished by attaching the C-terminal amino acid of thesequence to an insoluble support followed by sequential addition of theremaining amino acids in the sequence. Techniques for solid phasesynthesis are described by Barany & Merrifield, The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963,and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill., 1984. Proteins of greater length may be synthesizedby condensation of the amino and carboxyl termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxyl terminalend (such as by the use of the coupling reagent N,N′-dicylohexylcarbodimide) are well known in the art. Once an antibodymolecule has been produced, it may be purified by any method known inthe art for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigens Protein A or Protein G, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies or fragments thereof may be fused to heterologous polypeptidesequences (referred to herein as “tags”) described above or otherwiseknown in the art to facilitate purification.

Compositions and Therapeutic Methods

A method for preventing or treating cancer in a subject is alsodisclosed, the method comprising administering to the subject atherapeutically effective amount of at least one of the disclosedantibodies, an antigen binding fragment thereof, a nucleic acid encodingthe antibody, and/or a nucleic acid encoding the antigen bindingfragment, thereby preventing or treating cancer.

The disclosed antibodies can be cytotoxic to cancer cells.

Preferably, the cancer is leukaemia (such as Chronic LymphocyticLeukaemia (CLL), Acute Lymphoblastic Leukaemia (ALL), Mantle CellLeukaemia or Hairy Cell Leukaemia), pancreatic cancer, prostate cancer,colon cancer, bladder cancer, ovarian cancer, glioblastoma, testicularcancer, uterine cancer, adrenal cancer, breast cancer, lung cancer,melanoma, neuroblastoma, sarcoma, renal cancer. Furthermore, ROR1 isexpressed on a subset of cancer stem cells.

The present invention also relates to the disclosed isolated monoclonalantibodies and antigen binding fragments thereof for use in thetreatment or prevention of cancer. Further, the present invention alsorelates to use of the disclosed isolated monoclonal antibodies andantigen binding fragments thereof in the manufacture of a medicament forthe treatment or prevention of cancer.

Preferably, the cancer is leukaemia (such as Chronic LymphocyticLeukaemia (CLL), Acute Lymphoblastic Leukaemia (ALL), Mantle CellLeukaemia or Hairy Cell Leukaemia), pancreatic cancer, prostate cancer,colon cancer, bladder cancer, ovarian cancer, glioblastoma, testicularcancer, uterine cancer, adrenal cancer, breast cancer, lung cancer,melanoma, neuroblastoma, sarcoma, renal cancer. Furthermore, ROR1 isexpressed on a subset of cancer stem cells.

The cancer or tumour does not need to be completely eliminated for thecomposition to be effective. For example, a composition can reduce thetumour by a desired amount, for example by at least 10%, at least 20%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, as compared to the absence of thecomposition.

In another example, the subject can also be administered an effectiveamount of an additional agent, such as a chemotherapy agent. The methodscan include administration of one on more additional agents known in theart.

A therapeutically effective amount of a ROR1-specific antibody orantigen binding fragment (or the nucleic acid encoding the antibody orantigen binding fragment), or nucleic acid, will depend upon theseverity of the disease and the general state of the patient's health. Atherapeutically effective amount of the antibody can provide eithersubjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer. Asnoted above, these compositions can be administered in conjunction withanother therapeutic agent, either simultaneously or sequentially. Forany application, the antibody, antigen binding fragment, or nucleic acidencoding the antibody or antigen binding fragment can be combined withchemotherapy.

Single or multiple administrations of the compositions including theantibody, antigen binding fragment, or nucleic acid encoding theantibody or antigen binding fragment, that are disclosed herein, areadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of at least one of the antibodies disclosed hereinto effectively treat the patient. The dosage can be administered oncebut may be applied periodically until either a therapeutic result isachieved or until side effects warrant discontinuation of therapy. Inone example, a dose of the antibody is infused for thirty minutes everyother day. In this example, about one to about ten doses can beadministered, such as three or six doses can be administered every otherday. In a further example, a continuous infusion is administered forabout five to about ten days. The subject can be treated at regularintervals, such as monthly, until a desired therapeutic result isachieved. Generally, the dose is sufficient to treat or amelioratesymptoms or signs of disease without producing unacceptable toxicity tothe patient.

Compositions are further disclosed that include one or more of theantibodies that specifically bind a ROR1 polypeptide, or antigen bindingfragments of any of these antibodies, and nucleic acids encoding theseantibodies (and antigen binding fragments) that are disclosed herein ina carrier. The compositions can be prepared in unit dosage forms foradministration to a subject. The amount and timing of administration areat the discretion of the treating physician to achieve the desiredpurposes. The antibody and/or nucleic acid can be formulated forsystemic or local administration. In one example, the antibody and/ornucleic acid is formulated for parenteral administration, such asintravenous administration. In some embodiments, administration isintramuscular.

Active ingredients can also be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980). Specifically, liposomes containingthe immunogens or antibodies can be prepared by such methods asdescribed in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985);Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Pat.Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation timeare disclosed in U.S. Pat. No. 5,013,556. The everse-phase evaporationmethod can be used with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Polypeptides of the present invention can be conjugated to theliposomes as described, for example, in Martin et al., J. Biol. Chem.,257:286-288 (1982) via a disulfide interchange reaction.

The compositions for administration can include a solution of theantibody that specifically binds a ROR1 polypeptide, dissolved in apharmaceutically acceptable carrier, such as an aqueous carrier. Avariety of aqueous carriers can be used, for example, buffered salineand the like. These compositions may be sterilized by conventional, wellknown sterilization techniques. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of antibody in theseformulations can vary widely, and will be selected primarily based onfluid volumes, viscosities, body weight and the like in accordance withthe particular mode of administration selected and the subject's needs.In some embodiments, administration is intravenous.

Controlled-release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995). Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein, suchas a cytotoxin or a drug, as a central core. In microspheres thetherapeutic is dispersed throughout the particle. Particles,microspheres, and microcapsules smaller than about 1 μm are generallyreferred to as nanoparticles, nanospheres, and nanocapsules,respectively. Capillaries have a diameter of approximately 5 μm so thatonly nanoparticles are administered intravenously. Microparticles aretypically around 100 μm in diameter and are administered subcutaneouslyor intramuscularly. See, for example, Kreuter, J., Colloidal DrugDelivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y.,pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled DrugDelivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp.315-339, (1992).

Polymers can be used for ion-controlled release of the antibodycompositions disclosed herein. Various degradable and nondegradablepolymeric matrices for use in controlled drug delivery are known in theart (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, theblock copolymer, polaxamer 407, exists as a viscous yet mobile liquid atlow temperatures but forms a semisolid gel at body temperature. It hasbeen shown to be an effective vehicle for formulation and sustaineddelivery of recombinant interleukin-2 and urease (Johnston et al.,Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech.44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. JPharm. 112:215-224, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa. (1993)).

A typical pharmaceutical composition for intravenous administrationincludes about 0.1 to 10 mg/kg of antibody per day, or 0.5 to 15 mg/kgof antibody per day. Dosages from 0.1 up to about 100 mg/kg per subjectper day may be used, particularly if the agent is administered to asecluded site and not into the circulatory or lymph system, such as intoa body cavity or into a lumen of an organ. Exemplary doses include 1 to10 mg/kg, such as 2 to 8 mg/kg, such as 3 to 6 mg/kg. Actual methods forpreparing administrable compositions will be known or apparent to thoseskilled in the art and are described in more detail in such publicationsas Remington's Pharmaceutical Science, 19th ed., Mack PublishingCompany, Easton, Pa. (1995).

Antibodies may be provided in lyophilized form and rehydrated withsterile water before administration, although they are also provided insterile solutions of known concentration. The antibody solution is thenadded to an infusion bag containing 0.9% sodium chloride, USP, andtypically administered at a dosage of from 0.1 to 10 mg/kg or 0.5 to 15mg/kg of body weight. Exemplary doses include 1 to 10 mg/kg, such as 2to 8 mg/kg, such as 3 to 6 mg/kg. Considerable experience is availablein the art in the administration of antibody drugs, which have beenmarketed in the U.S. since the approval of RITUXAN® in 1997. Antibodiescan be administered by slow infusion, rather than in an intravenous pushor bolus. In one example, a higher loading dose is administered, withsubsequent, maintenance doses being administered at a lower level. Forexample, an initial loading dose of 4 mg/kg may be infused over a periodof some 90 minutes, followed by weekly maintenance doses for 4-8 weeksof 2 mg/kg infused over a 30 minute period if the previous dose was welltolerated.

A therapeutically effective amount of a nucleic acid encoding theantibody or an antigen binding fragment thereof can be administered to asubject in need thereof. One approach to administration of nucleic acidsis direct immunization with plasmid DNA, such as with a mammalianexpression plasmid. The nucleotide sequence encoding the antibody orfragment thereof can be placed under the control of a promoter toincrease expression of the molecule. Immunization by nucleic acidconstructs is well known in the art and taught, for example, in U.S.Pat. Nos. 5,643,578, and 5,593,972 and 5,817,637. U.S. Pat. No.5,880,103 describes several methods of delivery of nucleic acids to anorganism. The methods include liposomal delivery of the nucleic acids.

In another approach to using nucleic acids, an antibody or antigenbinding fragment thereof can also be expressed by attenuated viral hostsor vectors or bacterial vectors, which can be administered to a subject.Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus,retrovirus, cytomegalovirus, poxvirus or other viral vectors can be usedto express the antibody. For example, vaccinia vectors are described inU.S. Pat. No. 4,722,848. BCG (Bacillus Calmette Guerin) provides anothervector for expression of the disclosed antibodies (see Stover, Nature351:456-460, 1991).

In one embodiment, a nucleic acid encoding the antibody or an antigenbinding fragment thereof is introduced directly into cells. For example,the nucleic acid can be loaded onto gold microspheres by standardmethods and introduced into the skin by a device such as Bio-Rad'sHeliosa Gene Gun. The nucleic acids can be “naked,” consisting ofplasmids under control of a strong promoter.

Typically, the DNA is injected into muscle, although it can also beinjected directly into other sites. Dosages for injection are usuallyaround 0.5 mg/kg to about 50 mg/kg, and typically are about 0.005 mg/kgto about 5 mg/kg (see, e.g., U.S. Pat. No. 5,589,466).

In some examples, a subject is administered the DNA encoding theantibody or antibody binding fragments thereof to provide in vivoantibody production, for example using the cellular machinery of thesubject. Immunization by nucleic acid constructs is well known in theart and taught, for example, in U.S. Pat. Nos. 5,643,578, and 5,593,972and 5,817,637. U.S. Pat. No. 5,880,103 describes several methods ofdelivery of nucleic acids encoding to an organism. The methods includeliposomal delivery of the nucleic acids. Such methods can be applied tothe production of an antibody, or antibody binding fragments thereof, byone of ordinary skill in the art.

One approach to administration of nucleic acids is direct administrationwith plasmid DNA, such as with a mammalian expression plasmid. Thenucleotide sequence encoding the disclosed antibody, or antibody bindingfragments thereof, can be placed under the control of a promoter toincrease expression.

Diagnostic Methods and Kits

A method of detecting cancer in a subject is disclosed, the methodcomprising:

-   -   contacting a biological sample from the subject with at least        one disclosed isolated monoclonal antibody or an antigen binding        fragment thereof, and detecting antibody bound to the sample,

wherein the presence of antibody bound to the sample indicates that thesubject has cancer.

Preferably the antibody specifically binds a ROR1 polypeptide, and thepresence of antibody bound to the sample indicates that the subject hasleukaemia (such as Chronic Lymphocytic Leukaemia (CLL), AcuteLymphoblastic Leukaemia (ALL), Mantle Cell Leukaemia or Hairy CellLeukaemia), pancreatic cancer, prostate cancer, colon cancer, bladdercancer, ovarian cancer, glioblastoma, testicular cancer, uterine cancer,adrenal cancer, breast cancer, lung cancer, melanoma, neuroblastoma,sarcoma, renal cancer. Furthermore, ROR1 is expressed on a subset ofcancer stem cells.

Preferably, the isolated monoclonal antibody is directly labeled.

The method may further comprise contacting the sample with a secondantibody that specifically binds the isolated monoclonal antibody; and

detecting the binding of the second antibody,

wherein an increase in binding of the second antibody to the sample ascompared to binding of the second antibody to a control sample detectsthe presence of cancer in the subject.

The sample can be any sample, including, but not limited to, tissue frombiopsies, autopsies and pathology specimens. Biological samples alsoinclude sections of tissues, for example, frozen sections taken forhistological purposes. Biological samples further include body fluids,such as blood, serum, plasma, sputum, spinal fluid, nasopharyngealsecretions or urine.

Also disclosed is a method of detecting ROR1 comprising:

-   -   contacting a biological sample from a subject with at least one        disclosed isolated monoclonal antibody or an antigen binding        fragment thereof, and

detecting antibody bound to the sample.

The detection of ROR1 can be achieved, for example, by contacting asample to be tested, optionally along with a control sample, with theantibody under conditions that allow for formation of a complex betweenthe antibody and the polypeptide. Complex formation is then detected(e.g., using an ELISA). When using a control sample along with the testsample, complex is detected in both samples and any statisticallysignificant difference in the formation of complexes between the samplesis indicative of the presence of ROR1 in the test sample.

In some embodiments, an antibody is directly labeled with a detectablelabel. In another embodiment, the antibody that binds the ROR1 (thefirst antibody) is unlabeled and a second antibody or other moleculethat can bind the antibody that binds the ROR1 polypeptide is utilized.As is well known to one of skill in the art, a second antibody is chosenthat is able to specifically bind the specific species and class of thefirst antibody. For example, if the first antibody is a human IgG, thenthe secondary antibody may be an anti-human-lgG. Other molecules thatcan bind to antibodies include, without limitation, Protein A andProtein G, both of which are available commercially.

Suitable labels for the antibody or secondary antibody are describedabove.

The immunoassays and method disclosed herein can be used for a number ofpurposes. Kits for detecting a ROR1 polypeptide will typically comprisean antibody that binds a ROR1 polypeptide, for example, any of theantibodies disclosed herein. In some embodiments, an antibody fragment,such as an Fv fragment or a Fab is included in the kit. In a furtherembodiment, the antibody is labeled (for example, with a fluorescent,radioactive, or an enzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use. The instructional materials may be written, in anelectronic form (such as a computer diskette or compact disk) or may bevisual (such as video files). The kits may also include additionalcomponents to facilitate the particular application for which the kit isdesigned. Thus, for example, the kit may additionally contain means ofdetecting a label (such as enzyme substrates for enzymatic labels,filter sets to detect fluorescent labels, appropriate secondary labelssuch as a secondary antibody, or the like). The kits may additionallyinclude buffers and other reagents routinely used for the practice of aparticular method. Such kits and appropriate contents are well known tothose of skill in the art.

In one embodiment, the diagnostic kit comprises an immunoassay. Althoughthe details of the immunoassays may vary with the particular formatemployed, the method of detecting the ROR1 polypeptide in a biologicalsample generally includes the steps of contacting the biological samplewith an antibody which specifically reacts, under immunologicallyreactive conditions, to the viral polypeptide. The antibody is allowedto specifically bind under immunologically reactive conditions to forman immune complex, and the presence of the immune complex (boundantibody) is detected directly or indirectly.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed. The examples should be read in combination with the figureswhich are as follows:

FIG. 1A-FIG. 1C: Identification of ROR1 binding domain: FIG. 1A: SUP-T1cells were transduced with retroviral vectors containing either the fullextracellular portion of ROR1 or only one or two extracellular domains.Non-transduced SUP-T1 (SUP-T1 NT) cells served as negative control. FIG.1B: All 12 chimeric antibodies and FIG. 1C all 10 positive clones inscFv format were incubated with SUP-T1 NT and the new stable cell linesat 4° C. for 30 min. Cells were washed and stained with a secondaryantibody (anti-human Fc-Dylight647), which was used as negative control.eROR1=extracellular ROR1, Ig-like=Immunoglobulin-like domain,Fz=Frizzled domain, Kg=Kringle domain.

FIG. 2A (clone A) and FIG. 2B (clone F): Humanization of rat scFvs: Atotal of 25 constructs per clone were generated by combining five VH andfive VL. All 25 scFv constructs were tested on ROR1⁺ and ROR1⁻ celllines. Secondary antibody alone and media alone served as negativecontrols, whilst the parental versions of each clone acted as positivecontrol.

FIG. 3 : K_(D) determination by surface plasmon resonance. Sensorgramsobtained using a Biacore X100 instrument are shown. Briefly, ROR1antibodies were immobilized using a CM5 chip and seven differentconcentrations of the Histidine-tagged ROR1 protein (extracellularregion) were injected. Concentrations ranged from 1.5 to 100 nm.

FIG. 4A-FIG. 4B: Complement-dependent cytotoxicity of ROR1 MAbs on CLLcells (n=3). FIG. 4A: Chimeric ROR1 MAbs supernatants were tested at 0.5ug/ml on CLL cells and PBMCs. Only clone A showed a significant toxicitycompared to the isotype (p<0.001). Rituximab (Rtx) was used as positivecontrol at 0.5 ug/ml and 10 ug/ml, and achieved significant cytotoxicityon CLL1 and CLL2 samples (p<0.001). Cytotoxicity on CLL samples wasnormalized to PBMCs' data. FIG. 4B: Cell surface staining for CD20 andROR1 by flow cytometry. The red area represents the isotype control.Error bars in FIG. 4A represent SD. Experiments were done intriplicates.

FIG. 5A-FIG. 5H: Epitope mapping of ROR1 clone A MAb. FIG. 5A-FIG. 5B:Reactivity of clone A with ROR1-derived overlapping peptides wasanalyzed by ELISA. FIG. 5C-FIG. 5H: Amino acid substitution within theepitope binding region was performed in ROR1-transduced cell lines.Also, the previously described clones R12, 4a5 and D10 were alsoincluded for comparison. Red circles indicate essential amino acids forantibody binding. One out of three experiments is shown. Experimentswere done in triplicates. Error bars represent SD.

FIG. 6A-FIG. 6D: Epitope mapping of ROR1 clone F MAb. FIG. 6A-FIG. 6B:Reactivity of clone F with ROR1-derived overlapping peptides wasanalyzed by ELISA. Since no linear epitope was identified, FIG. 6C-FIG.6D: amino acid substitution of non-conserved regions within the Fzdomain was performed in ROR1-Fz transduced cell lines. Circles indicateessential amino acids for antibody binding. One out of three experimentsis shown. Experiments were done in triplicates. Error bars represent SD.

FIG. 7A-FIG. 7C: Competition assay by flow cytometry. Clone A (SA1) inmurine IgG2a, k (mSA1) along with other ROR1 MAbs in human IgG1,k-including previously published 4a5, D10 and R12 clones—were used forstaining ROR1-transduced cells. MAb staining was done as FIG. 7A-FIG.7B: single agents or FIG. 7C: in combination with clone A (mSA1).Anti-human IgG (450) and anti-mouse IgG (DyLight649) were used assecondary antibodies. Combined staining using clones F and mSA1 acted asnegative control for overlapping epitopes.

FIG. 8A-FIG. 8C: Internalisation of ROR1 antibodies on SKW 6.4 GFP cellsby flow cytometry. SKW 6.4 GFP cells were stained at 4° C. and eitherkept on ice (blue line) or incubated at 37° C. (yellow line) for 1 h. Ofall tested MAbs, clone V showed a pronounced reduction in MFI (greencircle). Clones A, F and Mu (purple circle) were selected for furtherinvestigation. Clones R12 and 4a5 (in black) served as negative andpositive control, respectively. Anti-human Fc-Dylight 647 was used assecondary antibody and as staining control (red line). MFI=MedianFluorescence Intensity.

FIG. 9A-FIG. 9C: Internalisation of ROR1 MAbs on SKW 6.4 GFP and CLLcells by flow cytometry. Cells were stained at 4° C. and either left onice or incubated at 37° C. for 15 min, 1 h and 2 h. Cells were thenanalysed using an anti-human IgG as secondary antibody. FIG. 9A-FIG. 9B:Histograms and FIG. 9C: Trends over time showing the MFI reduction overtime are presented. The % of MFI reduction was calculated as describedin the Materials and Methods chapter. Phenylarsine oxide (PAO), anendocytosis inhibitor, acted as negative control (PAO-120).

EXAMPLES Example 1

Immunization of Rats and Production of ROR1 Antibodies

A total of 3 Wistar rats were immunized using a DNA-based protocol.Briefly, human ROR1 coding sequence cloned into an immunization plasmidwas introduced into the rats, the target protein was expressed and animmune response was generated. Four applications of DNA using a gene gunwere initially performed. Rat serum was then analyzed, followed by 4additional applications. After confirming that serum from all threechallenged rats showed presence of anti-human ROR1 antibodies by flowcytometry, animals were sacrificed after 102 days of immunization. Lymphnodes were removed and pooled in order to produce oligoclonal hybridomacell lines.

A total of 38 positive hybridomas were identified by testing theirbinding ability to cells transfected with either pB1-ROR1-hum or with anirrelevant construct. As before, this was assessed by flow cytometry.

Antibody sequences were obtained by 5′ Rapid Amplification of cDNA ends.Oligoclonal hybridomas from Aldevron GmBH were separated into singlecell clones either by dilution or single cell sorting into 96 wellplates and colonies grown until confluent (approximately 2 weeks).Supernatant was screened against ROR1 positive and negative cell linesto ensure the presence of a specific anti-ROR1 antibody and also usedfor isotyping using rat immunoglobulin isotyping kits (eBioscience or BDBioscience).

Clones were grown until confluence in 6 well plates or 10 cm plates andthen pelleted into RNAlater (Life Technologies) before RNA was extractedusing RNA MiniPlus Kit (Qiagen). RNA was reverse transcribed to cDNAusing Quantitect Reverse Transcriptase (Qiagen). An aliquot of this cDNAwas assessed with GAPDH primers which were able to differentiate genomicand cDNA to ensure quality of samples. cDNA had a polyC tail added withTerminal Transferase (New England Biolabs) and nested PCR reactions wereperformed (Phusion Taq; New England Biolabs or Platinum Taq HighFidelity: Life Technologies) to identify the variable regions of theheavy and light chains, using primers specific for light chain isotypeand heavy chain isotype.

PCR products were run on a 1% TBE gel and post-stained with Gelstar(Lonza). Bands of the correct size were extracted and sent for directsequencing or inserted into Topo subcloning vectors (Life Technologies)for subsequent sequencing.

Sequence data was compared to the IMGT V-QUEST database of Rat germlineimmunoglobulin sequences and consensus sequences obtained that wereproductive and had an in frame signal sequence (Brochet et al., 2008,Alamyar et al., 2012). Overlap extension primers were designed toamplify the heavy and light chains whilst introducing a linker sequenceto generate ScFv constructs. A secreted version of the ScFv was producedby cloning the ScFv sequence in frame with murine IgG2a constant regionusing NcoI and BamHI sites (or if needed the compatible BglII or BclIsites).

Antibodies were generated by cloning the variable sequence in frame withhuman IgG1 or mouse heavy IgG2a chain constant regions, and light chainwith the corresponding human or mouse kappa constant regions. NcoI andMluI sites (or if needed, the compatible BspHI or PciI sites) were used.For humanized antibodies, variable regions were cloned in frame withhuman heavy IgG1 and light kappa constant regions.

A total of 17 novel scFvs were generated. Out of these, 12 clones boundas antibodies and 10 bound in a single chain variable fragment (scFv)format (See Table 1). Additionally, to identify the binding domain ofall positive clones, stable cell lines expressing either the fullextracellular region of ROR1 (eROR1) or varying regions of itsextracellular domains: Ig-like alone, Ig-like+Frizzled (Fz), Fz alone,Fz+Kringle (Kg) and Kg alone were generated by transducing SUP-T1 cellswith retroviral supernatants (See FIG. 1A).

In FIGS. 1B and 1C, we present flow cytometry data showing the bindingdomains of all 12 antibodies and 10 scFvs, respectively.

TABLE 1 Binds as whole Binds in Binding Domain immunoglobulin scFvformat Clone G3 Ig Yes No Clone G5 Ig Yes Yes Clone E7 Ig Yes No Clone JIg Yes No Clone F Fz Yes Yes Clone B Ig Yes Yes Clone A Ig Yes Yes CloneI Ig Yes Yes Clone O Ig Yes Yes Clone Pi Ig Yes Yes Clone Mu Ig Yes YesClone R Ig Not tested Yes Clone V Between Ig and Fz Yes Yes

As can be seen in FIG. 1C, in addition to the 10 scFv clones, two knownROR1 antibodies (R12 and 4A5) were also tested.

With the exception of clone F, which binds the frizzled domain and cloneV, which binds only the Ig-Fz protein, all other clones bound theimmunoglobulin domain. Interestingly, and somewhat surprisingly, theprior art antibodies R12 and 4A5 show different and distinct bindingcharacteristics to Clone F.

Example 2

Humanisation of Rat scFvs

Clones A and F were selected for humanisation. The variable domainsequences of rat scFvs were searched against the human IgG germlinedatabase. Five human framework sequences with high homology to each ratantibody were chosen as human acceptors for both light and heavy chainsCDRs. The sequences of five humanized VLs and humanized VHs wereobtained after directly grafting the CDRs of each rat antibody to thehuman acceptor frameworks.

For each clone, a total of 25 constructs were generated by combining allfive VH and five VL. Cloning was performed as described above. Bindingwas then tested on ROR1⁺ and ROR1⁻ cell lines. Parental versions ofclone A and F scFvs served as positive control for antibody staining,whilst media alone and secondary antibody alone acted as negativecontrols (See FIG. 2 ).

Example 3

Binding Efficiencies

We undertook surface plasmon resonance evaluation using a Biacore X100instrument. ROR1 chimeric antibodies were immobilised using ananti-human IgG1 antibody capture kit and a CM5 sensor chip. Sevendifferent concentrations, ranging from 1.5-100 nM, of the extracellularportion of ROR1 bearing a Histidine tag were then injected.

Surface plasmon resonance analysis showed that clones B5, A and Pipossessed the strongest affinities (1.51, 1.81 and 1.98 nM,respectively), whilst clone F presented the weakest one (5.46 nM).

Table 2 below further illustrates the binding kinetics of our ROR1chimeric antibodies.

Affinity values (K_(D)) of chimeric ROR1 antibodies ROR1 AntibodyK_(on), (10⁵)M⁻¹s⁻¹ K_(off), (10⁻⁴)s⁻¹ K_(D), 10⁻⁹ M E7 4.168 8.4902.037 G3 1.086 2.660 2.450 G5 1.593 4.164 2.614 A 5.774 10.44 1.809 B54.639 6.980 1.505 F 1.531 8.362 5.460 J6 2.492 8.351 3.351 I 2.093 5.1002.436 Pi 1.763 3.487 1.979 O 1.735 6.075 3.501

Example 4

Anti-Human ROR1 Antibody Cytotoxicity on Chronic Lymphoid Leukemia (CLL)Cells—Complement-Dependent Cytotoxicity (CDC) Assay

A CDC assay of the antibodies described above was carried out. Twoantibodies from other groups, namely R12 and 4A5, were also tested tosee how the antibodies of the invention compared to previously publishedantibodies. Antibodies were generated with human IgG1 Fc domain. Theantibodies were incubated for two hours with CLL cells, after whichcytotoxicity on the cells was determined.

Antibodies were tested at a concentration of 0.5 ug/ml. The isotype andRituximab (Rtx) controls were therefore used at this concentration. ForRituximab however we used an additional concentration of 10 ug/ml inorder to have a true positive control for the assay.

In FIG. 4A, CDC activity elicited by all 12 of our own ROR1 chimericantibodies can be observed. Of all antibodies evaluated, clone A was theonly one that showed significant cytotoxicity compared to the isotype(p<0.001). Remarkably, clone A was also better at killing CLL cells thanthe known ROR1 antibodies R12 and 4a5 but was still not as CDC active asRituximab.

The expression levels of both CD20 and ROR1 antigens on the surface ofCLL samples was also assessed. FIG. 4B explains why Rituximab is able tokill CD20 expressing cells very efficiently (CLL1 and 2), but not CLL3,as the latter was virtually negative for CD20 expression.

Example 5

Epitope Mapping

The epitopes of clones A and F described above were mapped by twoapproaches: peptide library ELISA and amino acid substitution.

Binding of clones A and F on overlapping peptides, covering all threeextracellular domains of ROR1, was tested by ELISA. This approachallowed us to narrow down the epitope of clone A to a five amino acidregion within the Ig-like domain (FIGS. 5A and 5B).

Point mutations were then generated for the Ig-like domain of human ROR1at positions 47 to 51. The particular mutations used were N(47)F,I(48)Q, S(50)D and the substitution of the five amino acid sequenceNISSE (SEQ ID NO: 253) at residues 47 to 51 with the sequence FQDDL (SEQID NO: 254).

Retroviral vectors were used for transducing SUP-T1 cells andtransduction was tested by GFP expression using flow cytometry.

It was found that the N(47)F and the I(48)Q substitutions reduced orstopped the clone A antibody binding to ROR1-Ig like domain, whereas theS(50)D substitution did not seem to affect binding. Further, the fiveamino acid substitution also prevented antibody binding. Therefore, itseems that Asn-47 and Ile-48 are essential for antibody binding. It wasfound that this epitope is unique to clone A and there is no overlapwith previously published ROR1 clones (R12, 4a5 and D10, the prototypeof Cirmtuzumab). Results can be seen in FIGS. 5(C)-5(H).

For clone F, peptide library ELISA data showed that it was not possibleto detect any binding signal above background (FIGS. 6A and 6B),suggesting that this antibody does not bind to a linear sequence, ratherit might potentially recognize a conformational epitope. Point mutationswere therefore generated for the Fz domain of human ROR1 atnon-conserved regions (positions 254 and 261). The particular mutationsused were I(254)V and Q(261)H.

Retroviral vectors were used for transducing SUP-T1 cells andtransduction was tested by GFP expression using flow cytometry.

It was found that the Q(261)H substitution reduced or stopped the cloneF antibody binding to ROR1-Fz domain, whereas the I(254)V substitutiondid not seem to affect binding. Further, the combination of Q(261)H andI(254)V also prevented antibody binding. Therefore, it seems thatGln-261 is essential for antibody binding. Results can be seen in FIGS.6C and 6D.

Example 6

Competition Assay by Flow Cytometry

To further challenge our previous observations—whereby clone A had adistinct epitope that was not shared with other anti-ROR1 antibodies—wedecided to compare the binding of A to ROR1 with other relevant clonesthrough a flow cytometry-based competition assay.

Based on previous studies, we hypothesised that consecutive- and/orsimultaneous-staining of ROR1+ cells, using clone A in combination withother anti-ROR1 antibodies, would allow us to identify overlappingepitopes by flow cytometry analysis.

To this end, we fused the variable region of clone A to a mouse IgG2a,kappa constants following the same protocols discussed in previouschapters. All other chimeric antibodies were kept in their existingrat-human format. We needed to generate A in a distinct constant regionin order to allow simultaneous and specific detection of every pair ofclones we were testing. In other words, since we were investigatingunlabelled clones, a secondary antibody staining against clonespossessing different isotypes was required. Also, for ease of analysis,ROR1⁺GFP⁺ cells were used.

FIGS. 7A and 7B shows strong ROR1 binding when antibodies were used assingle agents, which confirmed their correct expression and detection ofROR1. Since A and F antibodies detect different extracellular domains,this combination was included in our studies as negative control foroverlapping epitopes.

In FIG. 7C, three different columns are presented. Dot plots on thefirst column correspond to ROR1 binding assessed when SUP-T1 ROR1 cellswere stained with clone A (SA1) (mIgG) in the first instance. After awashing step, cells were stained with a competitor antibody (hIgG),followed by another washing step. Both antibodies were then detected bya third staining step using the appropriate secondary antibodies. In thesecond column, a similar approach was taken when staining these cells,except that this time it was the antibody in hIgG1, k format the oneused for the first staining step, followed by clone A (SA1) staining. Onthe third column, both antibodies were used at the same time.

In all cases, a clear and defined shift of all events was observed;indicating that, independent of the order in which cells were stained,antibodies used in the first staining step did not impede binding of theantibodies used in the second step. This was further confirmed when bothantibodies were assessed at the same time. Hence, these results confirmthat clone A binds to a unique epitope within the Ig-like domain, whichis not shared (not even partially) with other reported clones.

Example 7

Assessment of Internalisation

Antibody internalisation can have important implications as it couldprovide us with the opportunity to develop armed antibodies that couldbe conjugated to toxic payloads, such as toxins, radioisotopes orchemotherapeutic agents. This class of therapeutic antibodies are calledAntibody-Drug Conjugates (ADCs). Interestingly, ADCs could have atherapeutic advantage over naked antibodies (unconjugated), in terms ofpotency and efficacy, as their cytotoxicity relies on the payload theycarry rather than the immune system of patients. Thus, we nextinvestigated whether our antibodies were able to get internalised intoROR1⁺ cells by flow cytometry and pH-Amine dye labelling.

By Flow Cytometry

SKW 6.4 cells, Epstein-Barr virus-transformed B cells endogenouslyexpressing comparable levels of ROR1 as CLL patients, were incubated onice with all 12 ROR1 chimeric antibodies. After 30 min, cells werewashed with ice-cold PBS, and either left on ice or incubated at 37° C.for 1 h. Subsequent staining with an anti-human Fc-Dylight 647 was usedto detect any primary antibody that had remained on the cell surface.Previously reported clones R12 and 4a5 were also included in this assayas negative and positive controls, respectively (FIG. 8 ).

From all tested antibodies, clone V (green circle) showed almostcomplete MFI reduction. A modest but detectable decrease in MFI levelswas observed in clones A and F (purple circles), similar to the onedetected for clone 4a5. As previously reported, clone R12 did not showsignificant MFI reduction after 1 h incubation at 37° C.

MFI reduction could be caused by dissociation or internalisation or acombination of both (15). In order to further investigate what wastriggering this drop in MFI for clones V, A and F, we decided to use anendocytosis inhibitor in our next experiments.

Endocytosis Inhibition

Phenylarsine oxide (PAO), a trivalent arsenical compound, is the typicalchemical chosen to block Clathryn-mediated endocytosis (CME), althoughit can also inhibit macropinocytosis and phagocytosis. CME is the beststudied mechanism of endocytosis, and it has been established thatreceptor tyrosine kinases (RTKs), such as ROR1, predominantly use thisform of intemalisation when engulfed by the cell membrane and drawninside the cell.

To distinguish between dissociation and internalisation, SKW 6.4 cellswere incubated on ice in the presence of our selected antibodies for 30min. Cells were then washed with ice-cold PBS and either left on ice orincubated at 37° C. for 15 min, 1 h or 2 h. For the 2 h time point, aduplicate sample was incubated with PAO (10 μM). Immediately afterincubation, all samples were washed with ice-cold PBS and stained withan anti-human Fc-Dylight 647 for 30 min. Cells were analysed by flowcytometry and MFI reduction was calculated.

Flow cytometry analysis showed that although clone V had an importantMFI reduction, it was mainly due to dissociation as PAO did notconsiderably block the drop in MFI after 2 h incubation at 37° C. Acombination of internalisation and dissociation was more evident for theother 3 clones, being internalisation the dominating factor for clone4a5. This was even more evident for clone A.

To verify these results, we repeated the experiment using SKW 6.4 cellsand 2 samples of primary cells from CLL patients, expressing either highor low levels of ROR1 (FIG. 9 ). Samples were processed as mentionedabove and analysed by flow cytometry. Interestingly, these dataconfirmed that whilst both dissociation and internalisation wereinvolved in the MFI reduction, for clone V dissociation was the mainreason for decrease in MFI. This was even clearer on CLL cells, wherevirtually no internalisation of clone V was detected.

The dissociation of clones F, 4a5 and A from the cell surface of CLLcells was very similar between samples and seemed to be independent ofROR1 levels. In this case, internalisation appeared to be the maincontributing factor to MFI reduction. A similar observation was detectedon SKW 6.4 cells; although on this cell model, clone A was the onlyantibody where MFI drop was almost completely blocked by PAO and onlypartially for clones F and 4a5, suggesting that although these last 3antibodies might get partially internalised, clone A might be the mostpromising one.

Example 8

Clone F is Unique to Other Antibodies Generated (Murine and Rabbit)Because of Sequence Homology

Human, murine, rabbit and rat ROR1 protein sequences were aligned usingUniprot web based software (http://www.uniprot.org/align/) and thevariation between the different species highlighted. Uniprot accessionnumbers: Human (Q01973), Murine (Q9Z139) and Rabbit (G1U5L1). For ratROR1, NCBI reference sequence NP_001102141.1 was used as thecorresponding Uniprot sequence was only partially complete.

Clone F binds to Q261, which was possible due to differences between ratand human amino acids at this position (the human amino acid at position261 is glutamine (Q) whereas the corresponding amino acid at thisposition in rat is histidine (H)). When rats are immunised with humanROR1, this amino acid difference is recognised as an immunogen relativeto the rat ROR1 sequence, against which an antibody is produced.

The known antibody R12 (rabbit) and murine ROR1 binders show homologywith human ROR1 at this site (i.e. they all have glutamine (Q) at thisposition). As a result, immunisation of rabbits or mice with human ROR1does not result in antibody production directed to this position as itis not immunogenic. In view of this, clone F is unique in its ability tobind to this epitope.

Example 9

Humanisation Imparts Advantages Compared to Non-Humanised ComparatorConstructs

One of the rationales for targeting ROR1 as opposed to CD19, is sparingof the normal ROR1 negative B cell population. However at the same time,continued presence of normal CD19+ B cells allows for immune responsesdirected against a rat derived scFv. This has been seen with murinescFvs and have led to clinically significant outcomes, includinganaphylaxis with mRNA modified mesothelin CAR T cells (Maus et al.,2013) or antibody responses, with α-folate receptor or carbonicanhydrase IX specific CAR T cells (Lamers et al., 2006, Kershaw et al.,2006). T cell mediated immune responses are also possible due to crosspresentation of components of the CAR on MHC. CD19 CAR T cells bycomparison, inherently diminish the risk of antibody based immuneresponses by eradicating the normal B cell population, with B cellrecurrence associated with a higher risk of relapse. By undertakinghumanisation, we have decreased the likelihood of immune responsesagainst the antibody leading to enhanced persistence and decreasedimmunogenicity.

SEQUENCE LISTING

The amino acid sequences listed are shown in the application usingstandard one letter codes for amino acids. The sequences below relate to13 clones (G3, G5, E7, J, F, B, A, I, O, Pi, Mu, R and V) that weredeveloped. Also contained in the table below are sequences relating tothe humanisation of clones A and F.

SEQ Sequence Clone Description ID NO: DVVMTQTPVSLPVSLGGQVSISCRSS G3Rat Light Chain Framework Region 1   1 QSLEHSNGDTF G3 Light Chain CDR1  2 LHWYLQKPGQSPRLLIY G3 Rat Light Chain Framework Region 2   3 RVS G3Light Chain CDR2   4 NRFSGVPDRFSGSGSGTDFTLKISRIEPEDLG G3Rat Light Chain Framework Region 3   5 DYYC LQSTHFPNT G3Light Chain CDR3   6 FGAGTKLELK G3 Rat Light Chain Framework Region 4  7 DIQLTQSPSTLSASLGERVTISCRAS G5 Rat Light Chain Framework Region 1   8QSISNS G5 Light Chain CDR1   9 LNWYQQKPDGTVKRLIY G5Rat Light Chain Framework Region 2  10 STS G5 Light Chain CDR2  11TLESGVPSRFSGSGSGTDFSLSISSLESEDFAM G5 Rat Light Chain Framework Region 3 12 YYC LQFATYPQVT G5 Light Chain CDR3  13 FGSGTKLEIK G5Rat Light Chain Framework Region 4  14 DIVLTQSPALAVSVGQRATISCRAS E7Rat Light Chain Framework Region 1  15 QSVSISRYNF E7 Light Chain CDR1 16 MHWYQQKPGQQPKLLIY E7 Rat Light Chain Framework Region 2  17 RAS E7Light Chain CDR2  18 NLASGIPARFSGSGSGTDFTLTINPVQADDIA E7Rat Light Chain Framework Region 3  19 TYYC QQNRESPRT E7Light Chain CDR3  20 FGGGTKLELK E7 Rat Light Chain Framework Region 4 21 DIVLTQSPALAVSVGQRATISCRAS J Rat Light Chain Framework Region 1  15QSVSISRYDF J Light Chain CDR1  22 MHWYQQKPGQQPKLLIY JRat Light Chain Framework Region 2  17 RAS J Light Chain CDR2  18NLASGIPARFSGSGSGTDFTLTINPVQADDIA J Rat Light Chain Framework Region 3 19 TYYC QQNRESPRT J Light Chain CDR3  20 FGGGTKLELK JRat Light Chain Framework Region 4  21 DIQMTQSPSFLSASVGDRVTINCKAS FRat Light Chain Framework Region 1  23 QNIDRY F Light Chain CDR1  24LNWYQQKLGEAPKRLLY F Rat Light Chain Framework Region 2  25 NTN FLight Chain CDR2  26 KLQTGIPSRFSGSGSATDFTLTISSLQPEDFAT FRat Light Chain Framework Region 3  27 YFC LQYNSLPLT F Light Chain CDR3 28 FGSGTKLEIK F Rat Light Chain Framework Region 4  14DIQMTQSPSSMSASLGDRVTFTCQAS A Rat Light Chain Framework Region 1  29QDIGNN A Light Chain CDR1  30 LIWFQQKPGKSPRPLMY ARat Light Chain Framework Region 2  31 FAT A Light Chain CDR2  32SLANGVPSRFSGSRSGSDYSLTISSLESEDLAD A Rat Light Chain Framework Region 3 33 YHC LQYREYPLT A Light Chain CDR3  34 FGSGTKLDLK ARat Light Chain Framework Region 4  35 DIRMTQSPASLSASLGETVTIECLTS BRat Light Chain Framework Region 1  36 EDIYSD B Light Chain CDR1  37LAWFQQKPGKSPQLLIY B Rat Light Chain Framework Region 2  38 DAN BLight Chain CDR2  39 SLQNGVPSRFGGCGSGTQYSLQISSLQSEDV BRat Light Chain Framework Region 3  40 ATYFC QQYKNYPPT BLight Chain CDR3  41 FGGGTKLVLK B Rat Light Chain Framework Region 4  42DIQLTQSPSSMSASLGDRVSLTCQSS I Rat Light Chain Framework Region 1  43QGIGKY I Light Chain CDR1  44 LSWYQHKPGKPPKAMIY IRat Light Chain Framework Region 2  45 YAT I Light Chain CDR2  46KLADGVPSRFSGSRSGSDFSLTISSLESEDIAIY I Rat Light Chain Framework Region 3 47 YC LQFDDYPWT I Light Chain CDR3  48 FGGGTKLELK IRat Light Chain Framework Region 4  21 DIVLTQSPALAVSLEQRVTIACKTS ORat Light Chain Framework Region 1  49 QNVDNHGISY O Light Chain CDR1  50MHWYQQKSGQEPKLLIY O Rat Light Chain Framework Region 2  51 EGS OLight Chain CDR2  52 NLAVGIPARFSGSGSGTDFTLTIDPVEADDIE ORat Light Chain Framework Region 3  53 TYYC QQSKDDPRT O Light Chain CDR3 54 FGGGTKLELK O Rat Light Chain Framework Region 4  21QFTLTQPKSVSGSLRSTITIPCERS R Rat Light Chain Framework Region 1  55SGDIGDSY R Light Chain CDR1  56 VSWYQQHLGRPPINVIY RRat Light Chain Framework Region 2  57 ADD R Light Chain CDR2  58QRPSEVSDRFSGSIDSSSNSASLTITNLQMDD R Rat Light Chain Framework Region 3 59 EADYFC QSYDRNVDFNTV R Light Chain CDR3  60 FGGGTKVTVL RRat Light Chain Framework Region 4  61 DIQLTQSPSSLSASLGDRVSLTCQSS PiRat Light Chain Framework Region 1  62 QGIGKY Pi Light Chain CDR1  44LSWFQHKPGKPPKPVIN Pi Rat Light Chain Framework Region 2  63 YAT PiLight Chain CDR2  46 NLADGVPSRFSGRRSGSDFSLTISSLESEDTAI PiRat Light Chain Framework Region 3  64 YYC LQFDDFRWT Pi Light Chain CDR3 65 VGGGTKLELK Pi Rat Light Chain Framework Region 4  66QFTLTQPKSVSGSLRSTITIPCERS Mu Rat Light Chain Framework Region 1  55SGDIGDNY Mu Light Chain CDR1  67 VSWYQQHLGRPPINVIY MuRat Light Chain Framework Region 2  57 ADD Mu Light Chain CDR2  58QRPSEVSDRFSGSIDSSSNSASLTITNLQMDD Mu Rat Light Chain Framework Region 3 59 EADYFC QSFDSNFDIPV Mu Light Chain CDR3  68 FGGGTKLTVL MuRat Light Chain Framework Region 4  69 DIKMTQSPSFLSASVGDRVTINCKAS VRat Light Chain Framework Region 1  70 QNITRF V Light Chain CDR1  71LNWYQQELGEAPTLLIY V Rat Light Chain Framework Region 2  72 NTN VLight Chain CDR2  26 NLQTGIPSRFSGSGSGTDFTLTISSLQPEDVA VRat Light Chain Framework Region 3  73 TYFC LQHGSRPRT V Light Chain CDR3 74 FGGGTKLELK V Rat Light Chain Framework Region 4  21DVVMTQTPVSLPVSLGGQVSISCRSSQSLEH G3 Rat Light Chain Variable Region  75SNGDTFLHWYLQKPGQSPRLLIYRVSNRFSG VPDRFSGSGSGTDFTLKISRIEPEDLGDYYCLQSTHFPNTFGAGTKLELK DIQLTQSPSTLSASLGERVTISCRASQSISNSL G5Rat Light Chain Variable Region  76 NWYQQKPDGTVKRLIYSTSTLESGVPSRFSGSGSGTDFSLSISSLESEDFAMYYCLQFATYPQ VTFGSGTKLEIKDIVLTQSPALAVSVGQRATISCRASQSVSISRY E7 Rat Light Chain Variable Region  77NFMHWYQQKPGQQPKLLIYRASNLASGIPA RFSGSGSGTDFTLTINPVQADDIATYYCQQNRESPRTFGGGTKLELK DIVLTQSPALAVSVGQRATISCRASQSVSISRY JRat Light Chain Variable Region  78 DFMHWYQQKPGQQPKLLIYRASNLASGIPARFSGSGSGTDFTLTINPVQADDIATYYCQQN RESPRTFGGGTKLELKDIQMTQSPSFLSASVGDRVTINCKASQNIDR F Rat Light Chain Variable Region  79YLNWYQQKLGEAPKRLLYNTNKLQTGIPSRF SGSGSATDFTLTISSLQPEDFATYFCLQYNSLPLTFGSGTKLEIK DIQMTQSPSSMSASLGDRVTFTCQASQDIG ARat Light Chain Variable Region  80 NNLIWFQQKPGKSPRPLMYFATSLANGVPSRFSGSRSGSDYSLTISSLESEDLADYHCLQYRE YPLTFGSGTKLDLKDIRMTQSPASLSASLGETVTIECLTSEDIYSDL B Rat Light Chain Variable Region  81AWFQQKPGKSPQLLIYDANSLQNGVPSRFG GCGSGTQYSLQISSLQSEDVATYFCQQYKNYPPTFGGGTKLVLK DIQLTQSPSSMSASLGDRVSLTCQSSQGIGK IRat Light Chain Variable Region  82 YLSWYQHKPGKPPKAMIYYATKLADGVPSRFSGSRSGSDFSLTISSLESEDIAIYYCLQFDDYP WTFGGGTKLELKDIVLTQSPALAVSLEQRVTIACKTSQNVDNH O Rat Light Chain Variable Region  83GISYMHWYQQKSGQEPKLLIYEGSNLAVGIP ARFSGSGSGTDFTLTIDPVEADDIETYYCQQSKDDPRTFGGGTKLELK QFTLTQPKSVSGSLRSTITIPCERSSGDIGDSY RRat Light Chain Variable Region  84 VSWYQQHLGRPPINVIYADDQRPSEVSDRFSGSIDSSSNSASLTITNLQMDDEADYFCQSYD RNVDFNTVFGGGTKVTVLDIQLTQSPSSLSASLGDRVSLTCQSSQGIGKYL Pi Rat Light Chain Variable Region  85SWFQHKPGKPPKPVINYATNLADGVPSRFS GRRSGSDFSLTISSLESEDTAIYYCLQFDDFRWTVGGGTKLELK QFTLTQPKSVSGSLRSTITIPCERSSGDIGDNY MuRat Light Chain Variable Region  86 VSWYQQHLGRPPINVIYADDQRPSEVSDRFSGSIDSSSNSASLTITNLQMDDEADYFCQSFDS NFDIPVFGGGTKLTVLDIKMTQSPSFLSASVGDRVTINCKASQNITRF V Rat Light Chain Variable Region  87LNWYQQELGEAPTLLIYNTNNLQTGIPSRFS GSGSGTDFTLTISSLQPEDVATYFCLQHGSRPRTFGGGTKLELK EVQLQESGPGLVKPAQSLSLTCSVT G3Rat Heavy Chain Framework Region 1  88 GYSITNMYR G3 Heavy Chain CDR1  89WNWIRKFPGNKLEWMGY G3 Rat Heavy Chain Framework Region 2  90 INTAGST G3Heavy Chain CDR2  91 DYSPSLRGRVSITGDTSKNQFFLHLTSVTTED G3Rat Heavy Chain Framework Region 3  92 TATYYC AGFITNPFDF G3Heavy Chain CDR3  93 WGQGVMVTVSS G3 Rat Heavy Chain Framework Region 4 94 EVQVVESGGGLVQPGRSLKLSCVPS G5 Rat Heavy Chain Framework Region 1  95GFTFNNYW G5 Heavy Chain CDR1  96 MTWIRQAPGKAPEWVAS G5Rat Heavy Chain Framework Region 2  97 ISNTGGST G5 Heavy Chain CDR2  98FYPDSVRGRFSISRDNTKGTLYLHMTSLRSED G5 Rat Heavy Chain Framework Region 3 99 TATYYC IRNMDA G5 Heavy Chain CDR3 100 WGQGTSVTVSS G5Rat Heavy Chain Framework Region 4 101 GKLVESGGGLLKPGGSLKLSCVAS E7Rat Heavy Chain Framework Region 1 102 GFTFDKYW E7 Heavy Chain CDR1 103MHWVRQAPGKGLEWIAE E7 Rat Heavy Chain Framework Region 2 104 IEYDGTET E7Heavy Chain CDR2 105 NYAPSIKDRFTISRDNAKNTLYLQMSNVRSE E7Rat Heavy Chain Framework Region 3 106 DAATYFC TTEEMYTTDYYYGFAY E7Heavy Chain CDR3 107 WGQGTLVTVSS E7 Rat Heavy Chain Framework Region 4108 DVKLVESGGGLLKPGGSLKLSCVAS J Rat Heavy Chain Framework Region 1 109GFSFSKYW J Heavy Chain CDR1 110 MHWVRQAPGQGLEWIAE JRat Heavy Chain Framework Region 2 111 IEYDGTET J Heavy Chain CDR2 105NYAPSIKDRFTISRDNAKNTLYLQMSNVRFE J Rat Heavy Chain Framework Region 3 112DAATYFC TTEEMHTTDYYYGFAY J Heavy Chain CDR3 113 WGQGTLVTVSS JRat Heavy Chain Framework Region 4 108 EVQLVESGGGLVQPGRSLKLSCAAS FRat Heavy Chain Framework Region 1 114 GFIFSEHN F Heavy Chain CDR1 115MAWVRQAPKKGLEWVAT F Rat Heavy Chain Framework Region 2 116 ISDDGRNT FHeavy Chain CDR2 117 YYRDSMRGRFTISRENARSTLYLQLDSLRSED FRat Heavy Chain Framework Region 3 118 TATYYC ASHRYNLFDS FHeavy Chain CDR3 119 WGQGVMVTVSS F Rat Heavy Chain Framework Region 4 94 QVQLQQSGTELVKPASSVRISCKAS A Rat Heavy Chain Framework Region 1 120GYTLTTNY A Heavy Chain CDR1 121 MHWIRQQPGNGLEWIGW ARat Heavy Chain Framework Region 2 122 IYPGNGNT A Heavy Chain CDR2 123KFNHKFDGRTTLTADKSSSIVYMQLSSLTSED A Rat Heavy Chain Framework Region 3124 SAVYFC ARSDFDY A Heavy Chain CDR3 125 WGQGVMVTVSS ARat Heavy Chain Framework Region 4  94 DVQLEESGGGLVRPGRSLKLSCADS BRat Heavy Chain Framework Region 1 126 GVNFSNRG B Heavy Chain CDR1 127MAWVRQAPTKGLEWVAT B Rat Heavy Chain Framework Region 2 128 ISYDGRII BHeavy Chain CDR2 129 YYRDSVKGRFSISRENAKSTLYLQMDSLRSED BRat Heavy Chain Framework Region 3 130 TATYYC ARHPIAADWYFDF BHeavy Chain CDR3 131 WGPGTMVTVSS B Rat Heavy Chain Framework Region 4132 EVQLVESGGGSVQPGRSLKLSCAAS I Rat Heavy Chain Framework Region 1 133GFTFSDYN I Heavy Chain CDR1 134 MAWVRQAPKKGPEWVAT IRat Heavy Chain Framework Region 2 135 ITYDVHNA I Heavy Chain CDR2 136YYRDSVKGRFTISRDDAKSTLYLQMDSLRSED I Rat Heavy Chain Framework Region 3137 TATYFC ARPGAY I Heavy Chain CDR3 138 WGQGTLVTVSS IRat Heavy Chain Framework Region 4 108 QVRLLQSGAALVKPGASVKMSCKAS ORat Heavy Chain Framework Region 1 139 GYTFTDYW O Heavy Chain CDR1 140MSWVKQSHGKSLEWIGE O Rat Heavy Chain Framework Region 2 141 IYPNSGAT OHeavy Chain CDR2 142 NFNEKFKDKATLTVDRSTSTAYMELSRLTSED ORat Heavy Chain Framework Region 3 143 SAIYYC ARGFPNNYLSWFAY OHeavy Chain CDR3 144 WGQGTLVTVSS O Rat Heavy Chain Framework Region 4108 QIQLVQSGPELKKPGESVKISCKAS R Rat Heavy Chain Framework Region 1 145GYTFTNYG R Heavy Chain CDR1 146 MYWVKQAPGQGLQYMGW RRat Heavy Chain Framework Region 2 147 INTETGKP R Heavy Chain CDR2 148TYADDFKGRFVFFLETSASTAYLQINNLKNED R Rat Heavy Chain Framework Region 3149 MATYFC AREVKHGLFHWFAY R Heavy Chain CDR3 150 WGQGTLVTVSS RRat Heavy Chain Framework Region 4 108 EVQLVESGGGLVQPGRSLTLSCSAS PiRat Heavy Chain Framework Region 1 151 GFTFRDYN Pi Heavy Chain CDR1 152MAWVRQAPRKGLEWVAT Pi Rat Heavy Chain Framework Region 2 153 ISFDDYNT PiHeavy Chain CDR2 154 YYRDSVKGRFTISRDDAKSTLYLQMDSLRSED PiRat Heavy Chain Framework Region 3 155 TATYYC ARPGTY Pi Heavy Chain CDR3156 WGQGTLVTVSS Pi Rat Heavy Chain Framework Region 4 108QVQLQQSGAELVKPGSSVRISCKAS Mu Rat Heavy Chain Framework Region 1 157GYTITSYD Mu Heavy Chain CDR1 158 MHWIKQQPGNGLEGIGW MuRat Heavy Chain Framework Region 2 159 IHPGNGKI Mu Heavy Chain CDR2 160KYNQKFNGKATLTVDKSSSTAYMQLSSLTSE Mu Rat Heavy Chain Framework Region 3161 DSAVYFC ARGTTRVFPWFAY Mu Heavy Chain CDR3 162 WGQGTLVTVSS MuRat Heavy Chain Framework Region 4 108 EVQLVESGGGLVQPGRSLKLSCAAS VRat Heavy Chain Framework Region 1 114 GFSFSNYG V Heavy Chain CDR1 163MHWIRQAPTKGLEWVAS V Rat Heavy Chain Framework Region 2 164 ISPTGGNT VHeavy Chain CDR2 165 YYRDSVKGRFTISRDNTKSTLYLQMDSLRSED VRat Heavy Chain Framework Region 3 166 TATYYC ATDDLYYSGPFAY VHeavy Chain CDR3 167 WGQGTLVTVSS V Rat Heavy Chain Framework Region 4108 EVQLQESGPGLVKPAQSLSLTCSVTGYSITN G3 Rat Heavy Chain Variable Region168 MYRWNWIRKFPGNKLEWMGYINTAGSTDY SPSLRGRVSITGDTSKNQFFLHLTSVTTEDTATYYCAGFITNPFDFWGQGVMVTVSS EVQVVESGGGLVQPGRSLKLSCVPSGFTFNN G5Rat Heavy Chain Variable Region 169 YWMTWIRQAPGKAPEWVASISNTGGSTFYPDSVRGRFSISRDNTKGTLYLHMTSLRSEDTA TYYCIRNMDAWGQGTSVTVSSGKLVESGGGLLKPGGSLKLSCVASGFTFDKY E7 Rat Heavy Chain Variable Region 170WMHWVRQAPGKGLEWIAEIEYDGTETNYA PSIKDRFTISRDNAKNTLYLQMSNVRSEDAATYFCTTEEMYTTDYYYGFAYWGQGTLVTVSS DVKLVESGGGLLKPGGSLKLSCVASGFSFSKY JRat Heavy Chain Variable Region 171 WMHWVRQAPGQGLEWIAEIEYDGTETNYAPSIKDRFTISRDNAKNTLYLQMSNVRFEDA ATYFCTTEEMHTTDYYYGFAYWGQGTLVTV SSEVQLVESGGGLVQPGRSLKLSCAASGFIFSEH F Rat Heavy Chain Variable Region 172NMAWVRQAPKKGLEWVATISDDGRNTYYR DSMRGRFTISRENARSTLYLQLDSLRSEDTATYYCASHRYNLFDSWGQGVMVTVSS QVQLQQSGTELVKPASSVRISCKASGYTLTT ARat Heavy Chain Variable Region 173 NYMHWIRQQPGNGLEWIGWIYPGNGNTKFNHKFDGRTTLTADKSSSIVYMQLSSLTSEDS AVYFCARSDFDYWGQGVMVTVSSDVQLEESGGGLVRPGRSLKLSCADSGVNFSN B Rat Heavy Chain Variable Region 174RGMAWVRQAPTKGLEWVATISYDGRIIYYR DSVKGRFSISRENAKSTLYLQMDSLRSEDTATYYCARHPIAADWYFDFWGPGTMVTVSS EVQLVESGGGSVQPGRSLKLSCAASGFTFSD IRat Heavy Chain Variable Region 175 YNMAWVRQAPKKGPEWVATITYDVHNAYYRDSVKGRFTISRDDAKSTLYLQMDSLRSEDT ATYFCARPGAYWGQGTLVTVSSQVRLLQSGAALVKPGASVKMSCKASGYTFT O Rat Heavy Chain Variable Region 176DYWMSWVKQSHGKSLEWIGEIYPNSGATN FNEKFKDKATLTVDRSTSTAYMELSRLTSEDSAIYYCARGFPNNYLSWFAYWGQGTLVTVSS QIQLVQSGPELKKPGESVKISCKASGYTFTNY RRat Heavy Chain Variable Region 177 GMYWVKQAPGQGLQYMGWINTETGKPTYADDFKGRFVFFLETSASTAYLQINNLKNEDM ATYFCAREVKHGLFHWFAYWGQGTLVTVSSEVQLVESGGGLVQPGRSLTLSCSASGFTFRD Pi Rat Heavy Chain Variable Region 178YNMAWVRQAPRKGLEWVATISFDDYNTYY RDSVKGRFTISRDDAKSTLYLQMDSLRSEDTATYYCARPGTYWGQGTLVTVSS QVQLQQSGAELVKPGSSVRISCKASGYTITSY MuRat Heavy Chain Variable Region 179 DMHWIKQQPGNGLEGIGWIHPGNGKIKYNQKFNGKATLTVDKSSSTAYMQLSSLTSEDSA VYFCARGTTRVFPWFAYWGQGTLVTVSSEVQLVESGGGLVQPGRSLKLSCAASGFSFSN V Rat Heavy Chain Variable Region 180YGMHWIRQAPTKGLEWVASISPTGGNTYYR DSVKGRFTISRDNTKSTLYLQMDSLRSEDTATYYCATDDLYYSGPFAYWGQGTLVTVSS QVQLVQSGAEVKKPGASVKVSCKAS AHumanised 1 Heavy Chain FR 1 181 MHWVRQAPGQRLEWMGW AHumanised 1 Heavy Chain FR 2 182 KFNHKFDGRVTITRDTSASTAYMELSSLRSED AHumanised 1 Heavy Chain FR 3 183 TAVYYC WGQGTLVTVSS AHumanised 1 Heavy Chain FR 4 108 QVQLVQSGAEVKKPGASVKVSCKAS AHumanised 2 Heavy Chain FR 1 181 MHWVRQAPGQGLEWMGW AHumanised 2 Heavy Chain FR 2 184 KFNHKFDGRVTMTRDTSTSTVYMELSSLRSE AHumanised 2 Heavy Chain FR 3 185 DTAVYYC WGQGTMVTVSS AHumanised 2 Heavy Chain FR 4 186 QVQLVQSGAEVKKPGASVKVSCKAS AHumanised 3 Heavy Chain FR 1 181 MHWVRQAPGQGLEWMGW AHumanised 3 Heavy Chain FR 2 184 KFNHKFDGRVTMTRDTSISTAYMELSRLRSD AHumanised 3 Heavy Chain FR 3 187 DTAVYYC WGQGTLVTVSS AHumanised 3 Heavy Chain FR 4 108 QVQLVQSGAEVKKPGSSVKVSCKAS AHumanised 4 Heavy Chain FR 1 188 MHWVRQAPGQGLEWMGW AHumanised 4 Heavy Chain FR 2 184 KFNHKFDGRVTITADKSTSTAYMELSSLRSED AHumanised 4 Heavy Chain FR 3 189 TAVYYC WGQGTLVTVSS AHumanised 4 Heavy Chain FR 4 108 QVQLVQSGSELKKPGASVKVSCKAS AHumanised 5 Heavy Chain FR 1 190 MHWVRQAPGQGLEWMGW AHumanised 5 Heavy Chain FR 2 184 KFNHKFDGRFVFSLDTSVSTAYLQISSLKAED AHumanised 5 Heavy Chain FR 3 191 TAVYYC WGQGTLVTVSS AHumanised 5 Heavy Chain FR 4 108 QVQLVQSGAEVKKPGASVKVSCKASGYTLTT AHumanised 1 Heavy Chain Variable 192 NYMHWVRQAPGQRLEWMGWIYPGNGNT RegionKFNHKFDGRVTITRDTSASTAYMELSSLRSED TAVYYCARSDFDYWGQGTLVTVSSQVQLVQSGAEVKKPGASVKVSCKASGYTLTT A Humanised 2 Heavy Chain Variable 193NYMHWVRQAPGQGLEWMGWIYPGNGNT Region KFNHKFDGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAISDFDYWGQGTMVTVSS QVQLVQSGAEVKKPGASVKVSCKASGYTLTT AHumanised 3 Heavy Chain Variable 194 NYMHWVRQAPGQGLEWMGWIYPGNGNT RegionKFNHKFDGRVTMTRDTSISTAYMELSRLRSD DTAVYYCARSDFDYWGQGTLVTVSSQVQLVQSGAEVKKPGSSVKVSCKASGYTLTT A Humanised 4 Heavy Chain Variable 195NYMHWVRQAPGQGLEWMGWIYPGNGNT Region KFNHKFDGRVTITADKSTSTAYMELSSLRSEDTAVYYCATSDFDYWGQGTLVTVSS QVQLVQSGSELKKPGASVKVSCKASGYTLTT AHumanised 5 Heavy Chain Variable 196 NYMHWVRQAPGQGLEWMGWIYPGNGNT RegionKFNHKFDGRFVFSLDTSVSTAYLQISSLKAED TAVYYCARSDFDYWGQGTLVTVSSDIQMTQSPSSLSASVGDRVTITCQAS A Humanised 1 Light Chain FR 1 197LIWFQQKPGKAPKSLIY A Humanised 1 Light Chain FR 2 198SLANGVPSRFSGSGSGTDFTLTISSLQPEDFA A Humanised 1 Light Chain FR 3 199 TYYCFGPGTKVDIK A Humanised 1 Light Chain FR 4 200 DIQMTQSPSAMSASVGDRVTITCQASA Humanised 2 Light Chain FR 1 201 LIWFQQKPGKVPKRLIY AHumanised 2 Light Chain FR 2 202 SLANGVPSRFSGSGSGTEFTLTISSLQPEDFAT AHumanised 2 Light Chain FR 3 203 YYC FGPGTKVDIK AHumanised 2 Light Chain FR 4 200 DIQMTQSPSSLSASVGDRVTITCQAS AHumanised 3 Light Chain FR 1 197 LIWYQQKPGKAPKRLIY AHumanised 3 Light Chain FR 2 204 SLANGVPSRFSGSGSGTEFTLTISSLQPEDFAT AHumanised 3 Light Chain FR 3 203 YYC FGQGTKLEIK AHumanised 3 Light Chain FR 4 205 DIQMTQSPSSLSASVGDRVTITCQAS AHumanised 4 Light Chain FR 1 197 LIWYQQKPGKAPKLLIY AHumanised 4 Light Chain FR 2 206 SLANGVPSRFSGSGSGTDFTFTISSLQPEDIAT AHumanised 4 Light Chain FR 3 207 YYC FGQGTKLEIK AHumanised 4 Light Chain FR 4 205 DIQMTQSPSSVSASVGDRVTITCQAS AHumanised 5 Light Chain FR 1 208 LIWYQQKPGKAPKLLIY AHumanised 5 Light Chain FR 2 206 SLANGVPSRFSGSGSGTDFTLTISSLQPEDFA AHumanised 5 Light Chain FR 3 199 TYYC FGQGTKVEIK AHumanised 5 Light Chain FR 4 209 DIQMTQSPSSLSASVGDRVTITCQASQDIGN AHumanised 1 Light Chain Variable 210 NLIWFQQKPGKAPKSLIYFATSLANGVPSRFSRegion GSGSGTDFTLTISSLQPEDFATYYCLQYREYPL TFGPGTKVDIKDIQMTQSPSAMSASVGDRVTITCQASQDIG A Humanised 2 Light Chain Variable 211NNLIWFQQKPGKVPKRLIYFATSLANGVPSR Region FSGSGSGTEFTLTISSLQPEDFATYYCLQYREYPLTFGPGTKVDIK DIQMTQSPSSLSASVGDRVTITCQASQDIGN AHumanised 3 Light Chain Variable 212 NLIWYQQKPGKAPKRLIYFATSLANGVPSRFSRegion GSGSGTEFTLTISSLQPEDFATYYCLQYREYPL TFGQGTKLEIKDIQMTQSPSSLSASVGDRVTITCQASQDIGN A Humanised 4 Light Chain Variable 213NLIWYQQKPGKAPKLLIYFATSLANGVPSRFS RegionGSGSGTDFTFTISSLQPEDIATYYCLQYREYPL TFGQGTKLEIKDIQMTQSPSSVSASVGDRVTITCQASQDIGN A Humanised 5 Light Chain Variable 214NLIWYQQKPGKAPKLLIYFATSLANGVPSRFS RegionGSGSGTDFTLTISSLQPEDFATYYCLQYREYPL TFGQGTKVEIK QVQLVESGGGVVQPGRSLRLSCAASF Humanised 1 Heavy Chain FR 1 215 MAWVRQAPGKGLEWVAT FHumanised 1 Heavy Chain FR 2 216 YYRDSMRGRFTISRDNSKNTLYLQMNSLRAE FHumanised 1 Heavy Chain FR 3 217 DTAVYYC WGQGTMVTVSS FHumanised 1 Heavy Chain FR 4 186 EVQLVESGGGLVQPGGSLRLSCAAS FHumanised 2 Heavy Chain FR 1 218 MAWVRQAPGKGLEWVST FHumanised 2 Heavy Chain FR 2 219 YYRDSMRGRFTISRDNSKNTLYLQMNSLRAE FHumanised 2 Heavy Chain FR 3 217 DTAVYYC WGQGTLVTVSS FHumanised 2 Heavy Chain FR 4 108 EVQLVESGGGLVQPGGSLRLSCAAS FHumanised 3 Heavy Chain FR 1 218 MAWVRQAPGKGLEWVAT FHumanised 3 Heavy Chain FR 2 216 YYRDSMRGRFTISRDNAKNSLYLQMNSLRAE FHumanised 3 Heavy Chain FR 3 220 DTAVYYC WGQGTMVTVSS FHumanised 3 Heavy Chain FR 4 186 EVQLVESGGGLVQPGGSLRLSCAAS FHumanised 4 Heavy Chain FR 1 218 MAWVRQAPGKGLVWVST FHumanised 4 Heavy Chain FR 2 221 YYRDSMRGRFTISRDNAKNTLYLQMNSLRA FHumanised 4 Heavy Chain FR 3 222 EDTAVYYC WGQGTLVTVSS FHumanised 4 Heavy Chain FR 4 108 EVQLVESGGGLVQPGRSLRLSCAAS FHumanised 5 Heavy Chain FR 1 223 MAWVRQAPGKGLEWVST FHumanised 5 Heavy Chain FR 2 219 YYRDSMRGRFTISRDNAKNSLYLQMNSLRAE FHumanised 5 Heavy Chain FR 3 224 DTALYYC WGQGTLVTVSS FHumanised 5 Heavy Chain FR 4 108 QVQLVESGGGVVQPGRSLRLSCAASGFIFSE FHumanised 1 Heavy Chain Variable 225 HNMAWVRQAPGKGLEWVATISDDGRNTY RegionYRDSMRGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCTSHRYNLFDSWGQGTMVTVSSEVQLVESGGGLVQPGGSLRLSCAASGFIFSEH F Humanised 2 Heavy Chain Variable 226NMAWVRQAPGKGLEWVSTISDDGRNTYYR Region DSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHRYNLFDSWGQGTLVTVSS EVQLVESGGGLVQPGGSLRLSCAASGFIFSEH FHumanised 3 Heavy Chain Variable 227 NMAWVRQAPGKGLEWVATISDDGRNTYY RegionRDSMRGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARHRYNLFDSWGQGTMVTVSSEVQLVESGGGLVQPGGSLRLSCAASGFIFSEH F Humanised 4 Heavy Chain Variable 228NMAWVRQAPGKGLVWVSTISDDGRNTYYR Region DSMRGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARHRYNLFDSWGQGTLVTVSS EVQLVESGGGLVQPGRSLRLSCAASGFIFSEH FHumanised 5 Heavy Chain Variable 229 NMAWVRQAPGKGLEWVSTISDDGRNTYYRRegion DSMRGRFTISRDNAKNSLYLQMNSLRAEDT ALYYCAKHRYNLFDSWGQGTLVTVSSDIQMTQSPSSLSASVGDRVTITCKAS F Humanised 1 Light Chain FR 1 230LNWYQQKPGKAPKRLIY F Humanised 1 Light Chain FR 2 231KLQTGVPSRFSGSGSGTEFTLTISSLQPEDFAT F Humanised 1 Light Chain FR 3 232 YYCFGQGTKLEIK F Humanised 1 Light Chain FR 4 205 DIQMTQSPSSLSASVGDRVTITCKASF Humanised 2 Light Chain FR 1 230 LNWFQQKPGKAPKSLIY FHumanised 2 Light Chain FR 2 233 KLQTGVPSKFSGSGSGTDFTLTISSLQPEDFA FHumanised 2 Light Chain FR 3 234 TYYC FGQGTRLEIK FHumanised 2 Light Chain FR 4 235 DIQMTQSPSSLSASVGDRVTITCKAS FHumanised 3 Light Chain FR 1 230 LNWYQQKPGKAPKLLIY FHumanised 3 Light Chain FR 2 236 KLQTGVPSRFSGSGSGTDFTLTISSLQPEDFA FHumanised 3 Light Chain FR 3 237 TYYC FGQGTKLEIK FHumanised 3 Light Chain FR 4 205 DIQLTQSPSFLSASVGDRVTITCKAS FHumanised 4 Light Chain FR 1 238 LNWYQQKPGKAPKLLIY FHumanised 4 Light Chain FR 2 236 KLQTGVPSRFSGSGSGTEFTLTISSLQPEDFAT FHumanised 4 Light Chain FR 3 232 YYC FGQGTKLEIK FHumanised 4 Light Chain FR 4 205 DIQMTQSPSTLSASVGDRVTITCKAS FHumanised 5 Light Chain FR 1 239 LNWYQQKPGKAPKLLIY FHumanised 5 Light Chain FR 2 236 KLQTGVPSRFSGSGSGTEFTLTISSLQPDDFA FHumanised 5 Light Chain FR 3 240 TYYC FGQGTKLEIK FHumanised 5 Light Chain FR 4 205 DIQMTQSPSSLSASVGDRVTITCKASQNIDR FHumanised 1 Light Chain Variable 241 YLNWYQQKPGKAPKRLIYNTNKLQTGVPSRRegion FSGSGSGTEFTLTISSLQPEDFATYYCLQYNSL PLTFGQGTKLEIKDIQMTQSPSSLSASVGDRVTITCKASQNIDR F Humanised 2 Light Chain Variable 242YLNWFQQKPGKAPKSLIYNTNKLQTGVPSKF Region SGSGSGTDFTLTISSLQPEDFATYYCLQYNSLPLTFGQGTRLEIK DIQMTQSPSSLSASVGDRVTITCKASQNIDR FHumanised 3 Light Chain Variable 243 YLNWYQQKPGKAPKLLIYNTNKLQTGVPSRFRegion SGSGSGTDFTLTISSLQPEDFATYYCLQYNSLP LTFGQGTKLEIKDIQLTQSPSFLSASVGDRVTITCKASQNIDRYL F Humanised 4 Light Chain Variable 244NWYQQKPGKAPKLLIYNTNKLQTGVPSRFS Region GSGSGTEFTLTISSLQPEDFATYYCLQYNSLPLTFGQGTKLEIK DIQMTQSPSTLSASVGDRVTITCKASQNIDR FHumanised 5 Light Chain Variable 245 YLNWYQQKPGKAPKLLIYNTNKLQTGVPSRFRegion SGSGSGTEFTLTISSLQPDDFATYYCLQYNSLP LTFGQGTKLEIK ARSDFDY AHumanised 1 Heavy Chain CDR3 125 AISDFDY A Humanised 2 Heavy Chain CDR3246 ARSDFDY A Humanised 3 Heavy Chain CDR3 125 ATSDFDY AHumanised 4 Heavy Chain CDR3 247 ARSDFDY A Humanised 5 Heavy Chain CDR3125 AXSDFDY (where X is R, I or T) A General Heavy Chain CDR3 248TSHRYNLFDS F Humanised 1 Heavy Chain CDR3 249 AKHRYNLFDS FHumanised 2 Heavy Chain CDR3 250 ARHRYNLFDS FHumanised 3 Heavy Chain CDR3 251 ARHRYNLFDS FHumanised 4 Heavy Chain CDR3 251 AKHRYNLFDS FHumanised 5 Heavy Chain CDR3 250 XXHRYNLFDS (where X₁ is A or T FGeneral Heavy Chain CDR3 252 and X₂ is S, K or R) NISSEROR1 epitope for clone A 253 FQDDL Substituted sequence for epitope 254mapping

The CDR sequences and the framework regions in the table above have beendetermined based on information on framework regions and CDRs from theIMGT (the international ImMunoGeneTics information system) database (seewww.imgt.org).

An alternative method for labelling CDRs is using the Kabat system andthis can give slightly different results. However, this can easily bedetermined by someone skilled in the art. For the avoidance of doubt,the CDR sequences in the variable regions based on the Kabat system areas follows, where the Kabat CDRs are in bold:

Clone G3 light chain variable region (SEQ ID NO: 75)DVVMTQTPVSLPVSLGGQVSISCRSSQSLEHSNGDTFLHWYLQKPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRIEPEDLGDYYCLQSTHFPNTFGAGTKLELK Clone G5 light chain variable region (SEQ ID NO: 76)DIQLTQSPSTLSASLGERVTISCRASQSISNSLNWYQQKPDGTVKRLIYSTSTLESGVPSRFSGSGSGTDFSLSISSLESEDFAMYYCLQFATYPQVTFGSGTKLEIK Clone E7 light chain variable region (SEQ ID NO: 77)DIVLTQSPALAVSVGQRATISCRASQSVSISRYNFMHWYQQKPGQQPKLLIYRASNLASGIPARFSGSGSGTDFTLTINPVQADDIATYYCQQNRESPRTFGGGTKLELK Clone J light chain variable region (SEQ ID NO: 78)DIVLTQSPALAVSVGQRATISCRASQSVSISRYDFMHWYQQKPGQQPKLLIYRASNLASGIPARFSGSGSGTDFTLTINPVQADDIATYYCQQNRESPRTFGGGTKLELK Clone F light chain variable region (SEQ ID NO: 79)DIQMTQSPSFLSASVGDRVTINCKASQNIDRYLNWYQQKLGEAPKRLLYNTNKLQTGIPSRFSGSGSATDFTLTISSLQPEDFATYFCLQYNSLPLTFGSGTKLEIK Clone A light chain variable region (SEQ ID NO: 80)DIQMTQSPSSMSASLGDRVTFTCQASQDIGNNLIWFQQKPGKSPRPLMYFATSLANGVPSRFSGSRSGSDYSLTISSLESEDLADYHCLQYREYPLTFGSGTKLDLK Clone B light chain variable region (SEQ ID NO: 81)DIRMTQSPASLSASLGETVTIECLTSEDIYSDLAWFQQKPGKSPQLLIYDANSLQNGVPSRFGGCGSGTQYSLQISSLQSEDVATYFCQQYKNYPPTFGGGTKLVLK Clone I light chain variable region (SEQ ID NO: 82)DIQLTQSPSSMSASLGDRVSLTCQSSQGIGKYLSWYQHKPGKPPKAMIYYATKLADGVPSRFSGSRSGSDFSLTISSLESEDIAIYYCLQFDDYPWTFGGGTKLELK Clone O light chain variable region (SEQ ID NO: 83)DIVLTQSPALAVSLEQRVTIACKTSQNVDNHGISYMHWYQQKSGQEPKLLIYEGSNLAVGIPARFSGSGSGTDFTLTIDPVEADDIETYYCQQSKDDPRTFGGGTKLELK Clone R light chain variable region (SEQ ID NO: 84)QFTLTQPKSVSGSLRSTITIPCERSSGDIGDSYVSWYQQHLGRPPINVIYADDQRPSEVSDRFSGSIDSSSNSASLTITNLQMDDEADYFCQSYDRNVDFNTVFGGGTKVTVL Clone Pi light chain variable region (SEQ ID NO: 85)DIQLTQSPSSLSASLGDRVSLTCQSSQGIGKYLSWFQHKPGKPPKPVINYATNLADGVPSRFSGRRSGSDFSLTISSLESEDTAIYYCLQFDDFRWTVGGGTKLELK Clone Mu light chain variable region (SEQ ID NO: 86)QFTLTQPKSVSGSLRSTITIPCERSSGDIGDNYVSWYQQHLGRPPINVIYADDQRPSEVSDRFSGSIDSSSNSASLTITNLQMDDEADYFCQSFDSNFDIPVFGGGTKLTVL Clone V light chain variable region (SEQ ID NO: 87)DIKMTQSPSFLSASVGDRVTINCKASQNITRFLNWYQQELGEAPTLLIYNTNNLQTGIPSRFSGSGSGTDFTLTISSLQPEDVATYFCLQHGSRPRTFGGGTKLELK Clone G3 heavy chain variable region (SEQ ID NO: 168)EVQLQESGPGLVKPAQSLSLTCSVTGYSITNMYRWNWIRKFPGNKLEWMGYINTAGSTDYSPSLRGRVSITGDTSKNQFFLHLTSVTTEDTATYYCAGFITNPFDFWGQGVMVTVSS Clone G5 heavy chain variable region (SEQ ID NO: 169)EVQVVESGGGLVQPGRSLKLSCVPSGFTFNNYWMTWIRQAPGKAPEWVASISNTGGSTFYPDSVRGRFSISRDNTKGTLYLHMTSLRSEDTATYYCIRNMDAWGQGTSVTVSS Clone E7 heavy chain variable region (SEQ ID NO: 170)GKLVESGGGLLKPGGSLKLSCVASGFTFDKYWMHWVRQAPGKGLEWIAEIEYDGTETNYAPSIKDRFTISRDNAKNTLYLQMSNVRSEDAATYFCTTEEMYTTDYYYGFAYWGQGTLVTVSS Clone J heavy chain variable region (SEQ ID NO: 171)DVKLVESGGGLLKPGGSLKLSCVASGFSFSKYWMHWVRQAPGQGLEWIAEIEYDGTETNYAPSIKDRFTISRDNAKNTLYLQMSNVRFEDAATYFCTTEEMHTTDYYYGFAYWGQGTLVTVSS Clone F heavy chain variable region (SEQ ID NO: 172)EVQLVESGGGLVQPGRSLKLSCAASGFIFSEHNMAWVRQAPKKGLEWVATISDDGRNTYYRDSMRGRFTISRENARSTLYLQLDSLRSEDTATYYCASHRYNLFDSWGQGVMVTVSS Clone A heavy chain variable region (SEQ ID NO: 173)QVQLQQSGTELVKPASSVRISCKASGYTLTTNYMHWIRQQPGNGLEWIGWIYPGNGNTKFNHKFDGRTTLTADKSSSIVYMQLSSLTSEDSAVYFCARSDFDYWGQGVMVTVSS Clone B heavy chain variable region (SEQ ID NO: 174)DVQLEESGGGLVRPGRSLKLSCADSGVNFSNRGMAWVRQAPTKGLEWVATISYDGRIIYYRDSVKGRFSISRENAKSTLYLQMDSLRSEDTATYYCARHPIAADWYFDFWGPGTMVTVSS Clone I heavy chain variable region (SEQ ID NO: 175)EVQLVESGGGSVQPGRSLKLSCAASGFTFSDYNMAWVRQAPKKGPEWVATITYDVHNAYYRDSVKGRFTISRDDAKSTLYLQMDSLRSEDTATYFCARPGAYWGQGTLVTVSS Clone O heavy chain variable region (SEQ ID NO: 176)QVRLLQSGAALVKPGASVKMSCKASGYTFTDYWMSWVKQSHGKSLEWIGEIYPNSGATNFNEKFKDKATLTVDRSTSTAYMELSRLTSEDSAIYYCARGFPNNYLSWFAYWGQGTLVTVSS Clone R heavy chain variable region (SEQ ID NO: 177)QIQLVQSGPELKKPGESVKISCKASGYTFTNYGMYWVKQAPGQGLQYMGWINTETGKPTYADDFKGRFVFFLETSASTAYLQINNLKNEDMATYFCAREVKHGLFHWFAYWGQGTLVTVSS Clone Pi heavy chain variable region (SEQ ID NO: 178)EVQLVESGGGLVQPGRSLTLSCSASGFTFRDYNMAWVRQAPRKGLEWVATISFDDYNTYYRDSVKGRFTISRDDAKSTLYLQMDSLRSEDTATYYCARPGTYWGQGTLVTVSS Clone Mu heavy chain variable region (SEQ ID NO: 179)QVQLQQSGAELVKPGSSVRISCKASGYTITSYDMHWIKQQPGNGLEGIGWIHPGNGKIKYNQKFNGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARGTTRVFPWFAYWGQGTLVTVSS Clone V heavy chain variable region (SEQ ID NO: 180)EVQLVESGGGLVQPGRSLKLSCAASGFSFSNYGMHWIRQAPTKGLEWVASISPTGGNTYYRDSVKGRFTISRDNTKSTLYLQMDSLRSEDTATYYCATDDLYYSGPFAYWGQGTLVTVSS Clone A Humanised 1 light chain variable region (SEQ ID NO: 210)DIQMTQSPSSLSASVGDRVTITCQASQDIGNNLIWFQQKPGKAPKSLIYFATSLANGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYREYPLTFGPGTKVDIK Clone A Humanised 2 light chain variable region (SEQ ID NO: 211)DIQMTQSPSAMSASVGDRVTITCQASQDIGNNLIWFQQKPGKVPKRLIYFATSLANGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYREYPLTFGPGTKVDIK Clone A Humanised 3 light chain variable region (SEQ ID NO: 212)DIQMTQSPSSLSASVGDRVTITCQASQDIGNNLIWYQQKPGKAPKRLIYFATSLANGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYREYPLTFGQGTKLEIK Clone A Humanised 4 light chain variable region (SEQ ID NO: 213)DIQMTQSPSSLSASVGDRVTITCQASQDIGNNLIWYQQKPGKAPKLLIYFATSLANGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYREYPLTFGQGTKLEIK Clone A Humanised 5 light chain variable region (SEQ ID NO: 214)DIQMTQSPSSVSASVGDRVTITCQASQDIGNNLIWYQQKPGKAPKLLIYFATSLANGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYREYPLTFGQGTKVEIK Clone A Humanised 1 heavy chain variable region (SEQ ID NO: 192)QVQLVQSGAEVKKPGASVKVSCKASGYTLTTNYMHWVRQAPGQRLEWMGWIYPGNGNTKFNHKFDGRVTITRDTSASTAYMELSSLRSEDTAVYYCARSDFDYWGQGTLVTVSS Clone A Humanised 2 heavy chain variable region (SEQ ID NO: 193)QVQLVQSGAEVKKPGASVKVSCKASGYTLTTNYMHWVRQAPGQGLEWMGWIYPGNGNTKFNHKFDGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAISDFDYWGQGTMVTVSS Clone A Humanised 3 heavy chain variable region (SEQ ID NO: 194)QVQLVQSGAEVKKPGASVKVSCKASGYTLTTNYMHWVRQAPGQGLEWMGWIYPGNGNTKFNHKFDGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSDFDYWGQGTLVTVSS Clone A Humanised 4 heavy chain variable region (SEQ ID NO: 195)QVQLVQSGAEVKKPGSSVKVSCKASGYTLTTNYMHWVRQAPGQGLEWMGWIYPGNGNTKFNHKFDGRVTITADKSTSTAYMELSSLRSEDTAVYYCATSDFDYWGQGTLVTVSS Clone A Humanised 5 heavy chain variable region (SEQ ID NO: 196)QVQLVQSGSELKKPGASVKVSCKASGYTLTTNYMHWVRQAPGQGLEWMGWIYPGNGNTKFNHKFDGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARSDFDYWGQGTLVTVSS Clone F Humanised 1 light chain variable region (SEQ ID NO: 241)DIQMTQSPSSLSASVGDRVTITCKASQNIDRYLNWYQQKPGKAPKRLIYNTNKLQTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYNSLPLTFGQGTKLEIK Clone F Humanised 2 light chain variable region (SEQ ID NO: 242)DIQMTQSPSSLSASVGDRVTITCKASQNIDRYLNWFQQKPGKAPKSLIYNTNKLQTGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCLQYNSLPLTFGQGTRLEIK Clone F Humanised 3 light chain variable region (SEQ ID NO: 243)DIQMTQSPSSLSASVGDRVTITCKASQNIDRYLNWYQQKPGKAPKLLIYNTNKLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYNSLPLTFGQGTKLEIK Clone F Humanised 4 light chain variable region (SEQ ID NO: 244)DIQLTQSPSFLSASVGDRVTITCKASQNIDRYLNWYQQKPGKAPKLLIYNTNKLQTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYNSLPLTFGQGTKLEIK Clone F Humanised 5 light chain variable region (SEQ ID NO: 245)DIQMTQSPSTLSASVGDRVTITCKASQNIDRYLNWYQQKPGKAPKLLIYNTNKLQTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCLQYNSLPLTFGQGTKLEIK Clone F Humanised 1 heavy chain variable region (SEQ ID NO: 225)QVQLVESGGGVVQPGRSLRLSCAASGFIFSEHNMAWVRQAPGKGLEWVATISDDGRNTYYRDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTSHRYNLFDSWGQGTMVTVSS Clone F Humanised 2 heavy chain variable region (SEQ ID NO: 226)EVQLVESGGGLVQPGGSLRLSCAASGFIFSEHNMAWVRQAPGKGLEWVSTISDDGRNTYYRDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHRYNLFDSWGQGTLVTVSS Clone F Humanised 3 heavy chain variable region (SEQ ID NO: 227)EVQLVESGGGLVQPGGSLRLSCAASGFIFSEHNMAWVRQAPGKGLEWVATISDDGRNTYYRDSMRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHRYNLFDSWGQGTMVTVSS Clone F Humanised 4 heavy chain variable region (SEQ ID NO: 228)EVQLVESGGGLVQPGGSLRLSCAASGFIFSEHNMAWVRQAPGKGLVWVSTISDDGRNTYYRDSMRGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARHRYNLFDSWGQGTLVTVSS Clone F Humanised 5 heavy chain variable region (SEQ ID NO: 229)EVQLVESGGGLVQPGRSLRLSCAASGFIFSEHNMAWVRQAPGKGLEWVSTISDDGRNTYYRDSMRGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKHRYNLFDSWGQGTLVTVSS 

Therefore, the CDRs when determined using the Kabat system are asfollows:

SEQ Sequence Clone Description ID NO: RSSQSLEHSNGDTFL G3Light Chain CDR1 255 H RVSNRFS G3 Light Chain CDR2 256 LQSTHFPNT G3Light Chain CDR3   6 NMYRWN G3 Heavy Chain CDR1 257 YINTAGSTDYSPSLR G3Heavy Chain CDR2 258 G FITNPFDF G3 Heavy Chain CDR3 259 RASQSISNSLN G5Light Chain CDR1 260 STSTLES G5 Light Chain CDR2 261 LQFATYPQVT G5Light Chain CDR3  13 NYWMT G5 Heavy Chain CDR1 262 SISNTGGSTFYPDSV G5Heavy Chain CDR2 263 RG NMDA G5 Heavy Chain CDR3 264 RASQSVSISRYNFMH E7Light Chain CDR1 265 RASNLAS E7 Light Chain CDR2 266 QQNRESPRT E7Light Chain CDR3  20 KYWMH E7 Heavy Chain CDR1 267 EIEYDGTETNYAPSI E7Heavy Chain CDR2 268 KD EEMYTTDYYYGFAY E7 Heavy Chain CDR3 269RASQSVSISRYDFMH J Light Chain CDR1 270 RASNLAS J Light Chain CDR2 266QQNRESPRT J Light Chain CDR3  20 KYWMH J Heavy Chain CDR1 267EIEYDGTETNYAPSI J Heavy Chain CDR2 268 KD EEMHTTDYYYGFAY JHeavy Chain CDR3 271 KASQNIDRYLN F Light Chain CDR1 272 NTNKLQT FLight Chain CDR2 273 LQYNSLPLT F Light Chain CDR3  28 EHNMA FHeavy Chain CDR1 274 TISDDGRNTYYRDSM F Heavy Chain CDR2 275 RG HRYNLFDSF Heavy Chain CDR3 276 QASQDIGNNLI A Light Chain CDR1 277 FATSLAN ALight Chain CDR2 278 LQYREYPLT A Light Chain CDR3  34 TNYMH AHeavy Chain CDR1 279 WIYPGNGNTKFNHKF A Heavy Chain CDR2 280 DG SDFDY AHeavy Chain CDR3 281 LTSEDIYSDLA B Light Chain CDR1 282 DANSLQN BLight Chain CDR2 283 QQYKNYPPT B Light Chain CDR3  41 NRGMA BHeavy Chain CDR1 284 TISYDGRIIYYRDSV B Heavy Chain CDR2 285 KGHPIAADWYFDF B Heavy Chain CDR3 286 QSSQGIGKYLS I Light Chain CDR1 287YATKLAD I Light Chain CDR2 288 LQFDDYPWT I Light Chain CDR3  48 DYNMA IHeavy Chain CDR1 289 TITYDVHNAYYRDSV I Heavy Chain CDR2 290 KG PGAY IHeavy Chain CDR3 291 KTSQNVDNHGISYMH O Light Chain CDR1 292 EGSNLAV OLight Chain CDR2 293 QQSKDDPRT O Light Chain CDR3  54 DYWMS OHeavy Chain CDR1 294 EIYPNSGATNFNEKF O Heavy Chain CDR2 295 KDGFPNNYLSWFAY O Heavy Chain CDR3 296 ERSSGDIGDSYVS R Light Chain CDR1 297ADDQRPS R Light Chain CDR2 298 QSYDRNVDFNTV R Light Chain CDR3  60 NYGMYR Heavy Chain CDR1 299 WINTETGKPTYADDF R Heavy Chain CDR2 300 KGEVKHGLFHWFAY R Heavy Chain CDR3 301 QSSQGIGKYLS Pi Light Chain CDR1 287YATNLAD Pi Light Chain CDR2 302 LQFDDFRWT Pi Light Chain CDR3  65 DYNMAPi Heavy Chain CDR1 289 TISFDDYNTYYRDSV Pi Heavy Chain CDR2 303 KG PGTYPi Heavy Chain CDR3 304 ERSSGDIGDNYV Mu Light Chain CDR1 305 ADDQRPS MuLight Chain CDR2 298 QSFDSNFDIPV Mu Light Chain CDR3  68 SYDMH MuHeavy Chain CDR1 306 WIHPGNGKIKYNQKF Mu Heavy Chain CDR2 307 NGGTTRVFPWFAY Mu Heavy Chain CDR3 308 KASQNITRFLN V Light Chain CDR1 309NTNNLQT V Light Chain CDR2 310 LQHGSRPRT V Light Chain CDR3  74 NYGMH VHeavy Chain CDR1 311 SISPTGGNTYYRDSV V Heavy Chain CDR2 312 KGDDLYYSGPFAY V Heavy Chain CDR3 313 QASQDIGNNLI Humanised ALight Chain CDR1 277 FATSLAN Humanised A Light Chain CDR2 278 LQYREYPLTHumanised A Light Chain CDR3  34 TNYMH Humanised A Heavy Chain CDR1 279WIYPGNGNTKFNHKF Humanised A Heavy Chain CDR2 280 DG SDFDY Humanised AHeavy Chain CDR3 281 KASQNIDRYLN Humanised F Light Chain CDR1 272NTNKLQT Humanised F Light Chain CDR2 273 LQYNSLPLT Humanised FLight Chain CDR3  28 EHNMA Humanised F Heavy Chain CDR1 274TISDDGRNTYYRDSM Humanised F Heavy Chain CDR2 275 RG HRYNLFDS Humanised FHeavy Chain CDR3 276

1-46. (canceled)
 47. An isolated single chain Fv protein (scFv)comprising a light chain variable domain and a heavy chain variabledomain wherein the light chain variable domain comprises a light chaincomplementarity determining region (LCDR)1, an LCDR2 and an LCDR3, andwherein the heavy chain variable domain comprises a heavy chaincomplementarity determining region (HCDR)1, an HCDR2 and an HCDR3,wherein LCDR1 comprises an amino acid sequence set forth in SEQ ID NO:24; LCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 26;and LCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 28;HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 115;HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 117; andHCDR3 comprises an amino acid sequences set forth in SEQ ID NO: 252, orwherein the LCDR1 comprises the amino acid sequence set forth as SEQ IDNO: 272, the LCDR2 comprises the amino acid sequence set forth as SEQ IDNO: 273, the LCDR3 comprises the amino acid sequence set forth as SEQ IDNO: 28, the HCDR1 comprises the amino acid sequence set forth as SEQ IDNO: 274, the HCDR2 comprises the amino acid sequence set forth as SEQ IDNO: 275, and the HCDR3 comprises the amino acid sequence set forth asSEQ ID NO: 276; and wherein the scFv specifically binds to a ROR1polypeptide.
 48. An isolated scFv according to claim 47, wherein whenHCDR3 comprises an amino acid sequence having the sequence set forth asSEQ ID NO: 252, or the HCDR3 comprises an amino acid sequence as setforth in SEQ ID NO: 119, 249, 250 or
 251. 49. An isolated scFv accordingto claim 47, wherein: (a) the light chain variable domain comprises aLight Chain Framework Region (LCFR)1 comprising the amino acid sequenceas set forth as one of SEQ ID NO: 23, SEQ ID NO: 230, SEQ ID NO: 238 orSEQ ID NO: 239; an LCFR2 comprising the amino acid sequence as set forthas one of SEQ ID NO: 25, SEQ ID NO: 231, SEQ ID NO: 233 or SEQ ID NO:236; an LCFR3 comprising the amino acid sequence as set forth as one ofSEQ ID NO: 27, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 237 or SEQ IDNO: 240; and an LCFR4 comprising the amino acid sequence as set forth asone of SEQ ID NO: 14, SEQ ID NO: 205 or SEQ ID NO: 234, wherein theLight Chain Framework Regions may include up to 10 amino acidsubstitutions in the amino acid sequences as set forth above, or (b) theheavy chain variable domain comprises a Heavy Chain Framework Region(HCFR)1 comprising the amino acid sequence as set forth as one of SEQ IDNO: 114, SEQ ID NO: 215, SEQ ID NO: 218 or SEQ ID NO: 223; an HCFR2comprising the amino acid sequence as set forth as one of SEQ ID NO:116, SEQ ID NO: 216, SEQ ID NO: 219 or SEQ ID NO: 221; an HCFR3comprising the amino acid sequence as set forth as one of SEQ ID NO:118, SEQ ID NO: 217, SEQ ID NO: 220, SEQ ID NO: 222 or SEQ ID NO: 224;and an HCFR4 comprising the amino acid sequence as set forth as one ofSEQ ID NO: 94, SEQ ID NO: 186 or SEQ ID NO: 108, wherein the Heavy ChainFramework Regions may include up to 10 amino acid substitutions in theamino acid sequences as set forth above.
 50. An isolated scFv accordingto claim 47, wherein: (a) the light chain variable domain comprises theamino acid sequence as set forth as one of SEQ ID NO: 79, SEQ ID NO:241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244 or SEQ ID NO: 245,or an amino acid sequence having at least 90% identity to the specifiedamino acid sequences; or (b) the heavy chain variable domain comprisesthe amino acid sequence as set forth as one of SEQ ID NO: 172, SEQ IDNO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228 or SEQ ID NO:229, or an amino acid sequence having at least 90% identity to thespecified amino acid sequences.
 51. An isolated scFv comprising a lightchain variable domain and a heavy chain variable domain wherein: (a) thelight chain variable domain comprises the amino acid sequence as setforth as SEQ ID NO: 79 and the heavy chain variable domain comprises theamino acid sequence as set forth as SEQ ID NO: 172; (b) the light chainvariable domain comprises the amino acid sequence as set forth as SEQ IDNO: 241 and the heavy chain variable domain comprises the amino acidsequence as set forth as SEQ ID NO: 225; (c) the light chain variabledomain comprises the amino acid sequence as set forth as SEQ ID NO: 242and the heavy chain variable domain comprises the amino acid sequence asset forth as SEQ ID NO: 226; (d) the light chain variable domaincomprises the amino acid sequence as set forth as SEQ ID NO: 243 and theheavy chain variable domain comprises the amino acid sequence as setforth as SEQ ID NO: 227; (e) the light chain variable domain comprisesthe amino acid sequence as set forth as SEQ ID NO: 244 and the heavychain variable domain comprises the amino acid sequence as set forth asSEQ ID NO: 228; or (f) the light chain variable domain comprises theamino acid sequence as set forth as SEQ ID NO: 245 and the heavy chainvariable domain comprises the amino acid sequence as set forth as SEQ IDNO:
 229. 52. The isolated scFv of claim 47 or claim 51, wherein: (a) thescFv is a humanized scFv; (b) the scFv binds to an epitope of ROR1,wherein the epitope comprises amino acid Gln-261; (c) the scFv islabeled; or (d) the scFv is labeled, wherein the label is a fluorescent,an enzymatic, or a radioactive label.
 53. An isolated scFv fragment ofthe isolated scFv of claim 47 or claim
 51. 54. The isolated scFvfragment of claim 53, wherein: (a) the scFv fragment is labeled; or (b)the scFv fragment is labeled, wherein the label is a fluorescent, anenzymatic, or a radioactive label.
 55. The isolated scFv of claim 47 orclaim 51, wherein: (a) the isolated scFv or isolated scFv fragment isincluded in a bispecific antibody; or (b) the isolated scFv or isolatedscFv fragment is included in a chimeric antigen receptor (CAR).
 56. Acomposition comprising the scFv of claim 47 or claim 51, or an scFvfragment thereof, and a pharmaceutically acceptable carrier.
 57. Anisolated nucleic acid encoding the scFv of claim 47 or claim 51, or anscFv fragment thereof.
 58. An isolated host cell transformed with anucleic acid encoding the scFv of claim 47 or claim 51, or an scFvfragment thereof.
 59. A method of detecting cancer in a subjectcomprising: contacting a biological sample from the subject with atleast one isolated scFv of claim 47 or claim 51, or an scFv fragmentthereof; and detecting scFv bound to the sample, wherein the presence ofscFv bound to the sample indicates that the subject has cancer.
 60. Themethod of detecting cancer of claim 59, wherein: (a) the scFvspecifically binds a ROR1 polypeptide, and wherein the presence of scFvbound to the sample indicates that the subject has leukemia, pancreaticcancer, prostate cancer, colon cancer, bladder cancer, ovarian cancer,glioblastoma, testicular cancer, uterine cancer, adrenal cancer, breastcancer, lung cancer, melanoma, neuroblastoma, sarcoma or renal cancer;(b) the isolated scFv is directly labeled; or (c) the method furthercomprises: contacting the sample with a second antibody thatspecifically binds the isolated scFv; and detecting the binding of thesecond antibody, wherein an increase in binding of the second antibodyto the sample as compared to binding of the second antibody to a controlsample detects the presence of cancer in the subject.
 61. The method ofdetecting cancer of claim 60, wherein the leukemia is ChronicLymphocytic Leukaemia (CLL), Acute Lymphoblastic Leukaemia (ALL), MantleCell Leukaemia or Hairy Cell Leukaemia.
 62. A method of treating cancerin a subject, comprising administering to the subject a therapeuticallyeffective amount of at least one scFv of claim 47 or claim 51, or anscFv fragment thereof, thereby treating cancer.
 63. The method oftreating cancer of claim 62, wherein the cancer is leukemia, pancreaticcancer, prostate cancer, colon cancer, bladder cancer, ovarian cancer,glioblastoma, testicular cancer, uterine cancer, adrenal cancer, breastcancer, lung cancer, melanoma, neuroblastoma, sarcoma or renal cancer.64. The method of treating cancer of claim 63, wherein the leukemia isChronic Lymphocytic Leukaemia (CLL), Acute Lymphoblastic Leukaemia(ALL), Mantle Cell Leukaemia or Hairy Cell Leukaemia.
 65. A kitcomprising an scFv according to claim 47 or claim 51, or an scFvfragment thereof.
 66. The kit according to claim 65, wherein: (a) thescFv or scFv fragment is directly labelled; or (b) the kit furthercomprises an immunoassay.
 67. An isolated monoclonal antibody comprisinga light chain variable domain and a heavy chain variable domain whereinthe light chain variable domain comprises a light chain complementaritydetermining region (LCDR)1, an LCDR2 and an LCDR3, and wherein the heavychain variable domain comprises a heavy chain complementaritydetermining region (HCDR)1, an HCDR2 and an HCDR3, wherein LCDR1comprises an amino acid sequence set forth in SEQ ID NO: 30; LCDR2comprises an amino acid sequence set forth in SEQ ID NO: 32; and LCDR3comprises an amino acid sequence set forth in SEQ ID NO: 34; HCDR1comprises an amino acid sequence set forth in SEQ ID NO: 121; HCDR2comprises an amino acid sequence set forth in SEQ ID NO: 123; and HCDR3comprises an amino acid sequences set forth in SEQ ID NO: 248.